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Surface proteins of Streptococcus agalactiae and horizontal gene transfer
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International Journal of Medical Microbiology 294 (2004) 169–175 www.elsevier.de/ijmm
REVIEW
Surface proteins of Streptococcus agalactiae and horizontal gene transfer Gerd Bro¨ker, Barbara Spellerberg Department of Medical Microbiology and Hygiene, University of Ulm, Robert Koch Str. 8, D-89081 Ulm, Germany
Abstract Streptococcus agalactiae is responsible for serious infectious diseases in neonates, immuno-compromised adult patients and causes bovine mastitis in animal hosts. Genome sequencing projects revealed strong indications for horizontal gene transfer events leading to virulence acquisition and genetic diversity in this species. Bacterial surface proteins establish the first contact with host tissues and represent interesting targets for the exchange of virulence properties among different streptococci. This review will focus on horizontal gene transfer events in characterized S. agalactiae surface proteins, mobile genetic elements adjacent to the corresponding genes and will discuss potential mechanisms of transfer. r 2004 Elsevier GmbH. All rights reserved. Keywords: Surface protein; Recombination; Streptococcus; Mobile genetic element
Content Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 The alpha C protein family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 The scpB-lmb chromosomal region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 Genes encoded in the scpB-lmb region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Mobile genetic elements flanking the scpB-lmb region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Mobile genetic elements incorporated into the scpB-lmb region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Putative mechanisms of recombination events in S. agalactiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Corresponding author. Tel.: +49-731-5002-4615; fax:+49-731-5002-4619.
E-mail address: [email protected] (B. Spellerberg). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.06.018
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Introduction Streptococcus agalactiae (group B streptococcus, GBS) belongs to the group of pyogenic streptococci. Most species of this group are b-hemolytic on blood agar plates and can cause a wide range of infectious diseases in human and animal hosts. Phylogenetic analysis of 55 different streptococcal species shows that S. agalactiae clusters together with S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis (Facklam, 2002), while S. pyogenes is found in a closely related neighboring cluster. The presence of almost identical genes, mosaic genes and identical mobile genetic elements are indications of genetic recombination events among these streptococci. Genetic recombination and horizontal gene transfer play an important role for the evolution of bacterial diversity. As streptococcal surface proteins are crucial for the first contact between bacteria and host tissues, they represent interesting target genes for the exchange of virulence properties. The recent publication of several S. agalactiae genome sequences revealed the presence of 14 genomic islands. Two of these islands encode known and previously characterized surface proteins of S. agalactiae. The genes for the alpha C protein family are located on island number IV while the C5a peptidase and the Lmb protein are found on island XII (Glaser et al., 2002). Comparison of these surface proteins with respective proteins of closely related streptococcal species supports the hypothesis that the encoding genes were acquired through horizontal gene transfer events.
resulting in the evasion of antibody detection (Gravekamp et al., 1996). Regarding virulence properties these proteins have been implicated in the adhesion and invasion of S. agalactiae to epithelial cells (Bolduc et al., 2002). Deletion of the alpha C protein gene results in reduced virulence in animal models (Li et al., 1997). Comparison of Alp3 that is present in serotpye V S. agalactiae strains with S. pyogenes surface proteins revealed that Alp3 is nearly identical to R28 of S. pyogenes, except for the number of tandem repeats (Lachenauer et al., 2000). R28 of S. pyogenes is an immunogenic surface protein that promotes binding of S. pyogenes to human epithelial cells and appears to have a chimeric structure derived from the surface proteins Rib, and the Alpha and Beta component of the S. agalactiae C protein (Stalhammar-Carlemalm et al., 1999). A similar mosaic structure was observed for the S. agalactiae Alp2 and Alp3 proteins (Lachenauer et al., 2000), suggesting incorporation of genetic material originating from diverse sources. Analysis of the chromosomal nucleotide sequences surrounding the alpha C protein genes showed the presence of insertion sequence elements IS1381 and IS1191, but could not account for potential mechanisms of horizontal gene transfer between S. pyogenes and S. agalactiae. Based on their observations Lachenauer et al. (2000) suggested that a common gene pool exists for hemolytic streptococci, a situation resembling findings for neisserial species (Maiden et al., 1996) and for streptococci of the viridans group (Reichmann et al., 1997).
The scpB-lmb chromosomal region The alpha C protein family The C protein complex was among the first surface proteins of S. agalactiae to be characterized. It can induce protective antibodies in a mouse animal model (Lancefield et al., 1975). Later, genetic studies revealed that the C protein complex consists of two independently expressed genes coding for the alpha and beta C protein component (Jerlstrom et al., 1991; Michel et al., 1992). The alpha component can be found in the majority of serotypes and strains. The S. agalactiae surface proteins alpha C, Rib, Alp2 and Alp3 show striking similarities in their genetic structure and immunologic crossreactivity, apparently constituting a common protein family of immunogenic group B streptococcal surface proteins, called alpha C protein family. In protein electrophoresis these surface proteins display a characteristic laddering phenomenon. The corresponding genes contain multiple tandem repeats and the number of repeats is important for recognition through the host immune system. Deletion of repeats is used as a mechanism to create antigenic diversity,
Our group has been working on a genetic region of S. agalactiae that displays the structure of a composite transposon. It is flanked by insertion sequence elements and contains the genes for known and putative pathogenicity factors such as the C5a peptidase (ScpB) and the Lmb surface protein (Franken et al., 2001) (Fig. 1A). We were able to show that this region is present in all of the human S. agalactiae strains we studied. We also found that similar structures exist in human S. dysgalactiae subspecies equisimilis strains (human group C and group G streptococci), while they are uncommon in animal isolates of closely related species (animal group C and group G streptococci). The strong association of lmb and scpB genes with the human origin of a S. agalactiae isolate and the absence of the genes in many bovine isolates was also seen in independent studies conducted by other groups (Yildirim et al., 2002; Dmitriev et al., 2002). Very striking was the high conservation of the genes encoded in this region between different species of b-hemolytic streptococci. Comparison of the nucleotide sequence of scpB and lmb
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Fig. 1. Graphic representation of the chromosomal region spanning from the upstream copy of ISSag2 to the downstream copy of this element in S. agalactiae strain 2603V/R. (A) Linear form found in the chromosome. (B) Postulated cyclic form. a The numbering refers to the position of homologous parts found in the tnpA gene from S. pyogenes (AF026542). b The annotation of the gene fragments in S. agalactiae refers to the nucleotide position of strain 2603V/3. *The box representing GBSi1 is present only in a subset of S. agalactiae strains. Some strains harbor the IS element IS1548 in this location or no mobile genetic element at all.
to the respective homologues of S. pyogenes and S. dysgalactiae subsp. equisimilis strains showed identities of approximately 98%.
Genes encoded in the scpB-lmb region Lmb has first been identified by our group as a surface protein that mediates binding of S. agalactiae to immobilized human laminin (Spellerberg et al., 1999). It is a 34-kDa lipoprotein that resembles members of the Lra I family. Members of this family have initially been characterized as adhesins in streptococcal species of the viridans group (Jenkinson, 1994). The corresponding genes were subsequently shown to encode the substrate binding protein of manganese transport operons in several streptococcal species (Kolenbrander et al., 1998; Paik et al., 2003; Dintilhac et al., 1997). However, the chromosomal region surrounding Lmb in S. agalactiae does not harbor any transport genes and Lmb mutants did not show altered growth kinetics in manganesedeficient broth (Spellerberg et al., 1999). Very close homologues of Lmb exist in S. pyogenes and S. dysgalactiae subsp. equisimilis (human group C and group G streptococci). The S. pyogenes homologue (Lsp/Lbp) has been shown to directly interact with
laminin (Terao et al., 2002) and to be involved in the binding of S. pyogenes with epithelial cells (Terao et al., 2002; Elsner et al., 2002). The function of the S. dysgalactiae subspecies equisimilis homologues has not been characterized yet. The scpB gene encodes the group B streptococcal C5a peptidase, a 126-kDa serine protease that is anchored to the peptidoglycan of the cell wall by an LPTXG motif (Chmouryguina et al., 1996). It was first identified in 1988 for its ability to cleave the chemotactic complement component C5a and thus to interfere with the recruitment of leukocytes to sites of infection (Hill et al., 1988). Its role as a virulence factor can be demonstrated in animal experiments that confirm the in vitro results of C5a peptidase effects on leukocyte recruitment. The ability to cleave C5a is host specific, since murine C5a preparations are not susceptible to cleavage (Bohnsack et al., 1997). Conflicting studies exist concerning the ability to cleave bovine C5a. However, as many bovine strains do not harbor a C5a peptidase its function is clearly not required for the ability of S. agalactiae to cause bovine mastitis. Apart from the enzymatic function of the protein, it has recently been shown to bind to fibronectin and to be crucial for the invasion of S. agalactiae into epithelial cells (Cheng et al., 2002). Based on the high conservation of the nucleotide sequence of the S. agalactiae and S. pyogenes C5a
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peptidase a genetic exchange between the two species has already been postulated in 1996 (Chmouryguina et al., 1996).
Mobile genetic elements flanking the scpB-lmb region Analysing the genomic region surrounding scpB and lmb we found that the region is flanked by two copies of the insertion sequence element ISSag2 (Franken et al., 2001). This element belongs to the IS 3 family, that is characterized by two open reading frames (orfB and orfA) coding for the transposase by translation frame shifting between orfA and orfB (Mahillon and Chandler, 1998). ISSag2 displays the typical features of this family including a DDE motif that constitutes the catalytic center of the transposase. A very close homologue of ISSag2 exists in S. dysgalactiae strains and has been designated ISSdy1 (Vasi et al., 2000). All of the S. agalactiae strains that harbor scpB and lmb, harbor the complete composite transposon structure with flanking copies of ISSag2. S. agalactiae strains with more than two copies of ISSag2 have not yet been described. However, multiple copies of this element are commonly found in several other streptococcal species of the pyogenic group. While ISSag2 homologues cannot be found in S. pyogenes, human and animal strains of S. dysgalactiae and S. canis strains harbor multiple copies of ISSag2 homologues (Franken et al., 2001). Another IS element ISSag1 is encoded in the composite transposon structure and found in different streptococcal species. The structure of ISSag1 resembles IS 3 family members. ISSag1 in S. agalactiae is only found as a single copy and always associated with the described composite transposon structure (Franken et al., 2004). The gene encoding the transposase is disrupted by a stop codon in all of the S. agalactiae strains we studied. Thus it seems unlikely that it represents a functional IS element. Nearly identical homologues of this element can be found in other streptococcal strains. Multiple copies are present in animal strains of S. dysgalactiae and in these strains the regular orfA and orfB encoding the transposase are present and not disrupted by a frame-shift mutation. Moreover S. dysgalactiae subsp. dysgalactiae is until now the only species of pyogenic streptococci that has been described to harbor multiple copies of ISSag2 and ISSag1 homologues. In addition to these findings, mosaic structures of both elements are present in some S. dysgalactiae subsp. dysgalactiae strains. This indicates that in S. dysgalactiae subsp. dysgalactiae recombination events occurred between the IS elements that are flanking the scpB and lmb genes in S. agalactiae. As
S. dysgalactiae subsp. dysgalactiae and bovine S. agalactiae isolates share the same ecological niche (bovine milk), recombination events between these two species seem possible and might have been crucial for the assembly of the scpB-lmb transposon structure.
Mobile genetic elements incorporated into the scpB-lmb region The scpB-lmb chromosomal region appears to be an area of increased genetic plasticity. A number of different S. agalactiae strains either contain the IS element IS1548 or the group II intron GBSi1 incorporated between scpB and lmb (Fig. 1). The insertion element IS1548 was first identified in S. agalactiae endocarditis isolates mostly belonging to the serotype III and showing a hyaluronidase-negative phenotype (Granlund et al., 1998). This element is also present in other S. agalactiae serotypes and S. pyogenes strains. The presence of an IS element in different streptococcal species is not an unusual finding. While the insertion of IS1548 into the hyaluronidase gene of S. agalactiae was the first integration site to be described, it is not the most common. In all S. agalactiae isolates tested for the presence of IS1548 and in some S. pyogenes strains it was found downstream of the C5a peptidase gene (Granlund et al., 1998). IS1548 belongs to the ISAs1 family (Gustafson et al., 1994) and displays homologies to H-repeats associated with mobile genetic elements found in Escherichia coli (Zhao et al., 1993). A role for H-repeats in horizontal gene transfer and genetic recombination events has previously been suggested (Stroeher et al., 1995). Granlund et al. (1998) thus proposed that larger genomic rearrangements might have occurred in isolates harboring IS1548. The intergenic region between scpB and lmb appears to be a hot spot for integration, as another mobile genetic element, GBSi1 can be found in this locus. While IS1548 is inserted 9 bp upstream of the putative promoter for lmb, the insertion site for GBSi1 is located 88 bp further upstream (Granlund et al., 2001). In most GBS strains only one of these two elements is present which might indicate that they are present in different phylogenetic lineages of S. agalactiae. However, serotype II isolates in which both elements are present have also been identified (Takahashi et al., 2002). GBSi1 is a group II intron, a mobile genetic element that can transpose via an RNA intermediate. In most cases insertion occurs into specific DNA target sites in alleles lacking the intron, a process called retrohoming. Retrotransposition into novel (ectopic) sites is less frequent (Michel and Ferat, 1995). Group II introns were originally described in eukaryotic organisms, while they have subsequently been found in bacteria. Beside
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the insertion of GBSi1 between lmb and scpB two more GBSi1 loci have been identified (Luan et al., 2003). The second insertion site is located near the hemolysin gene cluster (Bohnsack et al., 2002). The third copy of GBSi1 is flanked by genes encoding a putative lipoate-protein ligase and a putative cobyric acid synthase (Luan et al., 2003). Detailed analysis of the area surrounding scpB in the S. agalactiae serotype III strain M732 by Luan et al. (2003) confirmed the presence of a composite transposon structure in this chromosomal region. The presence of a group II intron is consistent with a composite transposon, as group II introns incorporated into composite transposons have frequently been described (Martinez-Abarca and Toro, 2000).
Putative mechanisms of recombination events in S. agalactiae Nucleotide sequence data of whole genome sequencing indicates repeated and multiple events of horizontal gene transfer in streptococci as well as in other species and reveals an abundance of mobile genetic elements that can facilitate these events. Nevertheless molecular mechanisms of how b-hemolytic streptococci might exchange genetic information have not been fully elucidated. In contrast to streptococci of the viridans group b-hemolytic streptococcal species have not been shown to be naturally competent. However, comparison of the genome sequences of S. agalactiae and Streptococcus pneumoniae revealed that homologues of the genes required for S. pneumoniae competence exist in S. agalactiae (Glaser et al., 2002). Transposons and phages are both present in b-hemolytic streptococci and may represent common mechanisms of these species to transfer DNA fragments. As mentioned before, the genetic locus harboring scpB and lmb displays the structure of a composite transposon. Between the duplicated flanking IS elements, scattered fragments of a S. pyogenes transposase gene are found in several distinct areas (Fig. 1A). In the serotype III S. agalactiae strain M732, that Luan et al. analyzed, three larger fragments of a transposase gene are present. Comparing strain M732 with the serotype Ia derivative O90R they found that the region upstream of scpB harbors the insertion of a 2.1 kb fragment. The G/C content of this DNA fragment (29.7%) was notably lower than reported for the genome of S. agalactiae (35.6–35.6%) (Glaser et al., 2002; Tettelin et al., 2002). The first 605 nucleotides of this fragment showed 98% identity to the amino terminal encoding part of a putative transposase gene tnpA of S. pyogenes (McLaughlin et al., 1999). Comparison with two tnpA fragments further downstream that are present in all of the S. agalactiae strains analyzed thus far, show that all
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of these fragments may belong to a single disrupted transposase gene. However, the fragments are several kilobases apart and the order appears to be scrambled. We wanted to know if a hypothetical model could explain the S. agalactiae chromosomal structure of the serotype III M732 strain in this particular region. In fact the assumption of a circular structure, consisting of the scpB and lmb genes and the intact tnpA gene might explain these findings. All of the genes of the circle display a more than 95% nucleotide sequence identity with S. pyogenes genes. If in subsequent steps the mobile genetic elements ISSag1, ISSag2 and GBSi1 were incorporated into the circle, followed by integration of the whole structure into the S. agalactiae chromosome and duplication of the ISSag2 element, the resulting structure would indeed match the chromosomal region of the M732 serotype III strain (Fig. 1B). This however is only a hypothetical model that gives a theoretical explanation of the chromosomal S. agalactiae structure. The evolution of the particular region might of course have taken place differently.
Outlook Besides the recombination events described in this review, the genome sequence of S. agalactiae revealed a total of 14 genomic islands integrated into the S. agalactiae chromosome. Both of the regions described in this review are located on the identified islands. Compared to other streptococcal genomes S. agalactiae harbors a large number of chromosomal integrations. Based on these findings it has been proposed that S. agalactiae became a pathogen through successive acquisition of exogenous virulence factors (Glaser et al., 2002). Several of these islands encode the genes of not yet characterized putative surface proteins and virulence factors. Future analysis and characterization of these genes may significantly contribute to our understanding of how pathogens evolve.
Acknowledgements The work of Gerd Bro¨ker and Barbara Spellerberg was supported by DFG grant Sp511/5-1.
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Tettelin, H., Masignani, V., Cieslewicz, M.J., Eisen, J.A., Peterson, S., Wessels, M.R., Paulsen, I.T., Nelson, K.E., Margarit, I., Read, T.D., Madoff, L.C., Wolf, A.M., Beanan, M.J., Brinkac, L.M., Daugherty, S.C., DeBoy, R.T., Durkin, A.S., Kolonay, J.F., Madupu, R., Lewis, M.R., Radune, D., Fedorova, N.B., Scanlan, D., Khouri, H., Mulligan, S., Carty, H.A., Cline, R.T., Van Aken, S.E., Gill, J., Scarselli, M., Mora, M., Iacobini, E.T., Brettoni, C., Galli, G., Mariani, M., Vegni, F., Maione, D., Rinaudo, D., Rappuoli, R., Telford, J.L., Kasper, D.L., Grandi, G., Fraser, C.M., 2002. Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc. Natl. Acad. Sci. USA 99, 12391–12396. Vasi, J., Lindberg, M., Guss, B., 2000. A novel IS-like element frequently inserted in a putative virulence regulator in bovine mastitis isolates of Streptococcus dysgalactiae. Plasmid 44, 220–230. Yildirim, A.O., Lammler, C., Weiss, R., Kopp, P., 2002. Pheno- and genotypic properties of streptococci of serological group B of canine and feline origin. FEMS Microbiol. Lett. 212, 187–192. Zhao, S., Sandt, C.H., Feulner, G., Vlazny, D.A., Gray, J.A., Hill, C.W., 1993. Rhs elements of E. coli K-12: complex composites of shared and unique components that have different evolutionary histories. J. Bacteriol. 175, 2799–2808.
International Journal of Medical Microbiology 294 (2004) 169–175 www.elsevier.de/ijmm
REVIEW
Surface proteins of Streptococcus agalactiae and horizontal gene transfer Gerd Bro¨ker, Barbara Spellerberg Department of Medical Microbiology and Hygiene, University of Ulm, Robert Koch Str. 8, D-89081 Ulm, Germany
Abstract Streptococcus agalactiae is responsible for serious infectious diseases in neonates, immuno-compromised adult patients and causes bovine mastitis in animal hosts. Genome sequencing projects revealed strong indications for horizontal gene transfer events leading to virulence acquisition and genetic diversity in this species. Bacterial surface proteins establish the first contact with host tissues and represent interesting targets for the exchange of virulence properties among different streptococci. This review will focus on horizontal gene transfer events in characterized S. agalactiae surface proteins, mobile genetic elements adjacent to the corresponding genes and will discuss potential mechanisms of transfer. r 2004 Elsevier GmbH. All rights reserved. Keywords: Surface protein; Recombination; Streptococcus; Mobile genetic element
Content Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 The alpha C protein family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 The scpB-lmb chromosomal region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 Genes encoded in the scpB-lmb region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Mobile genetic elements flanking the scpB-lmb region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Mobile genetic elements incorporated into the scpB-lmb region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Putative mechanisms of recombination events in S. agalactiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Corresponding author. Tel.: +49-731-5002-4615; fax:+49-731-5002-4619.
E-mail address: [email protected] (B. Spellerberg). 1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2004.06.018
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Introduction Streptococcus agalactiae (group B streptococcus, GBS) belongs to the group of pyogenic streptococci. Most species of this group are b-hemolytic on blood agar plates and can cause a wide range of infectious diseases in human and animal hosts. Phylogenetic analysis of 55 different streptococcal species shows that S. agalactiae clusters together with S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis (Facklam, 2002), while S. pyogenes is found in a closely related neighboring cluster. The presence of almost identical genes, mosaic genes and identical mobile genetic elements are indications of genetic recombination events among these streptococci. Genetic recombination and horizontal gene transfer play an important role for the evolution of bacterial diversity. As streptococcal surface proteins are crucial for the first contact between bacteria and host tissues, they represent interesting target genes for the exchange of virulence properties. The recent publication of several S. agalactiae genome sequences revealed the presence of 14 genomic islands. Two of these islands encode known and previously characterized surface proteins of S. agalactiae. The genes for the alpha C protein family are located on island number IV while the C5a peptidase and the Lmb protein are found on island XII (Glaser et al., 2002). Comparison of these surface proteins with respective proteins of closely related streptococcal species supports the hypothesis that the encoding genes were acquired through horizontal gene transfer events.
resulting in the evasion of antibody detection (Gravekamp et al., 1996). Regarding virulence properties these proteins have been implicated in the adhesion and invasion of S. agalactiae to epithelial cells (Bolduc et al., 2002). Deletion of the alpha C protein gene results in reduced virulence in animal models (Li et al., 1997). Comparison of Alp3 that is present in serotpye V S. agalactiae strains with S. pyogenes surface proteins revealed that Alp3 is nearly identical to R28 of S. pyogenes, except for the number of tandem repeats (Lachenauer et al., 2000). R28 of S. pyogenes is an immunogenic surface protein that promotes binding of S. pyogenes to human epithelial cells and appears to have a chimeric structure derived from the surface proteins Rib, and the Alpha and Beta component of the S. agalactiae C protein (Stalhammar-Carlemalm et al., 1999). A similar mosaic structure was observed for the S. agalactiae Alp2 and Alp3 proteins (Lachenauer et al., 2000), suggesting incorporation of genetic material originating from diverse sources. Analysis of the chromosomal nucleotide sequences surrounding the alpha C protein genes showed the presence of insertion sequence elements IS1381 and IS1191, but could not account for potential mechanisms of horizontal gene transfer between S. pyogenes and S. agalactiae. Based on their observations Lachenauer et al. (2000) suggested that a common gene pool exists for hemolytic streptococci, a situation resembling findings for neisserial species (Maiden et al., 1996) and for streptococci of the viridans group (Reichmann et al., 1997).
The scpB-lmb chromosomal region The alpha C protein family The C protein complex was among the first surface proteins of S. agalactiae to be characterized. It can induce protective antibodies in a mouse animal model (Lancefield et al., 1975). Later, genetic studies revealed that the C protein complex consists of two independently expressed genes coding for the alpha and beta C protein component (Jerlstrom et al., 1991; Michel et al., 1992). The alpha component can be found in the majority of serotypes and strains. The S. agalactiae surface proteins alpha C, Rib, Alp2 and Alp3 show striking similarities in their genetic structure and immunologic crossreactivity, apparently constituting a common protein family of immunogenic group B streptococcal surface proteins, called alpha C protein family. In protein electrophoresis these surface proteins display a characteristic laddering phenomenon. The corresponding genes contain multiple tandem repeats and the number of repeats is important for recognition through the host immune system. Deletion of repeats is used as a mechanism to create antigenic diversity,
Our group has been working on a genetic region of S. agalactiae that displays the structure of a composite transposon. It is flanked by insertion sequence elements and contains the genes for known and putative pathogenicity factors such as the C5a peptidase (ScpB) and the Lmb surface protein (Franken et al., 2001) (Fig. 1A). We were able to show that this region is present in all of the human S. agalactiae strains we studied. We also found that similar structures exist in human S. dysgalactiae subspecies equisimilis strains (human group C and group G streptococci), while they are uncommon in animal isolates of closely related species (animal group C and group G streptococci). The strong association of lmb and scpB genes with the human origin of a S. agalactiae isolate and the absence of the genes in many bovine isolates was also seen in independent studies conducted by other groups (Yildirim et al., 2002; Dmitriev et al., 2002). Very striking was the high conservation of the genes encoded in this region between different species of b-hemolytic streptococci. Comparison of the nucleotide sequence of scpB and lmb
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Fig. 1. Graphic representation of the chromosomal region spanning from the upstream copy of ISSag2 to the downstream copy of this element in S. agalactiae strain 2603V/R. (A) Linear form found in the chromosome. (B) Postulated cyclic form. a The numbering refers to the position of homologous parts found in the tnpA gene from S. pyogenes (AF026542). b The annotation of the gene fragments in S. agalactiae refers to the nucleotide position of strain 2603V/3. *The box representing GBSi1 is present only in a subset of S. agalactiae strains. Some strains harbor the IS element IS1548 in this location or no mobile genetic element at all.
to the respective homologues of S. pyogenes and S. dysgalactiae subsp. equisimilis strains showed identities of approximately 98%.
Genes encoded in the scpB-lmb region Lmb has first been identified by our group as a surface protein that mediates binding of S. agalactiae to immobilized human laminin (Spellerberg et al., 1999). It is a 34-kDa lipoprotein that resembles members of the Lra I family. Members of this family have initially been characterized as adhesins in streptococcal species of the viridans group (Jenkinson, 1994). The corresponding genes were subsequently shown to encode the substrate binding protein of manganese transport operons in several streptococcal species (Kolenbrander et al., 1998; Paik et al., 2003; Dintilhac et al., 1997). However, the chromosomal region surrounding Lmb in S. agalactiae does not harbor any transport genes and Lmb mutants did not show altered growth kinetics in manganesedeficient broth (Spellerberg et al., 1999). Very close homologues of Lmb exist in S. pyogenes and S. dysgalactiae subsp. equisimilis (human group C and group G streptococci). The S. pyogenes homologue (Lsp/Lbp) has been shown to directly interact with
laminin (Terao et al., 2002) and to be involved in the binding of S. pyogenes with epithelial cells (Terao et al., 2002; Elsner et al., 2002). The function of the S. dysgalactiae subspecies equisimilis homologues has not been characterized yet. The scpB gene encodes the group B streptococcal C5a peptidase, a 126-kDa serine protease that is anchored to the peptidoglycan of the cell wall by an LPTXG motif (Chmouryguina et al., 1996). It was first identified in 1988 for its ability to cleave the chemotactic complement component C5a and thus to interfere with the recruitment of leukocytes to sites of infection (Hill et al., 1988). Its role as a virulence factor can be demonstrated in animal experiments that confirm the in vitro results of C5a peptidase effects on leukocyte recruitment. The ability to cleave C5a is host specific, since murine C5a preparations are not susceptible to cleavage (Bohnsack et al., 1997). Conflicting studies exist concerning the ability to cleave bovine C5a. However, as many bovine strains do not harbor a C5a peptidase its function is clearly not required for the ability of S. agalactiae to cause bovine mastitis. Apart from the enzymatic function of the protein, it has recently been shown to bind to fibronectin and to be crucial for the invasion of S. agalactiae into epithelial cells (Cheng et al., 2002). Based on the high conservation of the nucleotide sequence of the S. agalactiae and S. pyogenes C5a
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peptidase a genetic exchange between the two species has already been postulated in 1996 (Chmouryguina et al., 1996).
Mobile genetic elements flanking the scpB-lmb region Analysing the genomic region surrounding scpB and lmb we found that the region is flanked by two copies of the insertion sequence element ISSag2 (Franken et al., 2001). This element belongs to the IS 3 family, that is characterized by two open reading frames (orfB and orfA) coding for the transposase by translation frame shifting between orfA and orfB (Mahillon and Chandler, 1998). ISSag2 displays the typical features of this family including a DDE motif that constitutes the catalytic center of the transposase. A very close homologue of ISSag2 exists in S. dysgalactiae strains and has been designated ISSdy1 (Vasi et al., 2000). All of the S. agalactiae strains that harbor scpB and lmb, harbor the complete composite transposon structure with flanking copies of ISSag2. S. agalactiae strains with more than two copies of ISSag2 have not yet been described. However, multiple copies of this element are commonly found in several other streptococcal species of the pyogenic group. While ISSag2 homologues cannot be found in S. pyogenes, human and animal strains of S. dysgalactiae and S. canis strains harbor multiple copies of ISSag2 homologues (Franken et al., 2001). Another IS element ISSag1 is encoded in the composite transposon structure and found in different streptococcal species. The structure of ISSag1 resembles IS 3 family members. ISSag1 in S. agalactiae is only found as a single copy and always associated with the described composite transposon structure (Franken et al., 2004). The gene encoding the transposase is disrupted by a stop codon in all of the S. agalactiae strains we studied. Thus it seems unlikely that it represents a functional IS element. Nearly identical homologues of this element can be found in other streptococcal strains. Multiple copies are present in animal strains of S. dysgalactiae and in these strains the regular orfA and orfB encoding the transposase are present and not disrupted by a frame-shift mutation. Moreover S. dysgalactiae subsp. dysgalactiae is until now the only species of pyogenic streptococci that has been described to harbor multiple copies of ISSag2 and ISSag1 homologues. In addition to these findings, mosaic structures of both elements are present in some S. dysgalactiae subsp. dysgalactiae strains. This indicates that in S. dysgalactiae subsp. dysgalactiae recombination events occurred between the IS elements that are flanking the scpB and lmb genes in S. agalactiae. As
S. dysgalactiae subsp. dysgalactiae and bovine S. agalactiae isolates share the same ecological niche (bovine milk), recombination events between these two species seem possible and might have been crucial for the assembly of the scpB-lmb transposon structure.
Mobile genetic elements incorporated into the scpB-lmb region The scpB-lmb chromosomal region appears to be an area of increased genetic plasticity. A number of different S. agalactiae strains either contain the IS element IS1548 or the group II intron GBSi1 incorporated between scpB and lmb (Fig. 1). The insertion element IS1548 was first identified in S. agalactiae endocarditis isolates mostly belonging to the serotype III and showing a hyaluronidase-negative phenotype (Granlund et al., 1998). This element is also present in other S. agalactiae serotypes and S. pyogenes strains. The presence of an IS element in different streptococcal species is not an unusual finding. While the insertion of IS1548 into the hyaluronidase gene of S. agalactiae was the first integration site to be described, it is not the most common. In all S. agalactiae isolates tested for the presence of IS1548 and in some S. pyogenes strains it was found downstream of the C5a peptidase gene (Granlund et al., 1998). IS1548 belongs to the ISAs1 family (Gustafson et al., 1994) and displays homologies to H-repeats associated with mobile genetic elements found in Escherichia coli (Zhao et al., 1993). A role for H-repeats in horizontal gene transfer and genetic recombination events has previously been suggested (Stroeher et al., 1995). Granlund et al. (1998) thus proposed that larger genomic rearrangements might have occurred in isolates harboring IS1548. The intergenic region between scpB and lmb appears to be a hot spot for integration, as another mobile genetic element, GBSi1 can be found in this locus. While IS1548 is inserted 9 bp upstream of the putative promoter for lmb, the insertion site for GBSi1 is located 88 bp further upstream (Granlund et al., 2001). In most GBS strains only one of these two elements is present which might indicate that they are present in different phylogenetic lineages of S. agalactiae. However, serotype II isolates in which both elements are present have also been identified (Takahashi et al., 2002). GBSi1 is a group II intron, a mobile genetic element that can transpose via an RNA intermediate. In most cases insertion occurs into specific DNA target sites in alleles lacking the intron, a process called retrohoming. Retrotransposition into novel (ectopic) sites is less frequent (Michel and Ferat, 1995). Group II introns were originally described in eukaryotic organisms, while they have subsequently been found in bacteria. Beside
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the insertion of GBSi1 between lmb and scpB two more GBSi1 loci have been identified (Luan et al., 2003). The second insertion site is located near the hemolysin gene cluster (Bohnsack et al., 2002). The third copy of GBSi1 is flanked by genes encoding a putative lipoate-protein ligase and a putative cobyric acid synthase (Luan et al., 2003). Detailed analysis of the area surrounding scpB in the S. agalactiae serotype III strain M732 by Luan et al. (2003) confirmed the presence of a composite transposon structure in this chromosomal region. The presence of a group II intron is consistent with a composite transposon, as group II introns incorporated into composite transposons have frequently been described (Martinez-Abarca and Toro, 2000).
Putative mechanisms of recombination events in S. agalactiae Nucleotide sequence data of whole genome sequencing indicates repeated and multiple events of horizontal gene transfer in streptococci as well as in other species and reveals an abundance of mobile genetic elements that can facilitate these events. Nevertheless molecular mechanisms of how b-hemolytic streptococci might exchange genetic information have not been fully elucidated. In contrast to streptococci of the viridans group b-hemolytic streptococcal species have not been shown to be naturally competent. However, comparison of the genome sequences of S. agalactiae and Streptococcus pneumoniae revealed that homologues of the genes required for S. pneumoniae competence exist in S. agalactiae (Glaser et al., 2002). Transposons and phages are both present in b-hemolytic streptococci and may represent common mechanisms of these species to transfer DNA fragments. As mentioned before, the genetic locus harboring scpB and lmb displays the structure of a composite transposon. Between the duplicated flanking IS elements, scattered fragments of a S. pyogenes transposase gene are found in several distinct areas (Fig. 1A). In the serotype III S. agalactiae strain M732, that Luan et al. analyzed, three larger fragments of a transposase gene are present. Comparing strain M732 with the serotype Ia derivative O90R they found that the region upstream of scpB harbors the insertion of a 2.1 kb fragment. The G/C content of this DNA fragment (29.7%) was notably lower than reported for the genome of S. agalactiae (35.6–35.6%) (Glaser et al., 2002; Tettelin et al., 2002). The first 605 nucleotides of this fragment showed 98% identity to the amino terminal encoding part of a putative transposase gene tnpA of S. pyogenes (McLaughlin et al., 1999). Comparison with two tnpA fragments further downstream that are present in all of the S. agalactiae strains analyzed thus far, show that all
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of these fragments may belong to a single disrupted transposase gene. However, the fragments are several kilobases apart and the order appears to be scrambled. We wanted to know if a hypothetical model could explain the S. agalactiae chromosomal structure of the serotype III M732 strain in this particular region. In fact the assumption of a circular structure, consisting of the scpB and lmb genes and the intact tnpA gene might explain these findings. All of the genes of the circle display a more than 95% nucleotide sequence identity with S. pyogenes genes. If in subsequent steps the mobile genetic elements ISSag1, ISSag2 and GBSi1 were incorporated into the circle, followed by integration of the whole structure into the S. agalactiae chromosome and duplication of the ISSag2 element, the resulting structure would indeed match the chromosomal region of the M732 serotype III strain (Fig. 1B). This however is only a hypothetical model that gives a theoretical explanation of the chromosomal S. agalactiae structure. The evolution of the particular region might of course have taken place differently.
Outlook Besides the recombination events described in this review, the genome sequence of S. agalactiae revealed a total of 14 genomic islands integrated into the S. agalactiae chromosome. Both of the regions described in this review are located on the identified islands. Compared to other streptococcal genomes S. agalactiae harbors a large number of chromosomal integrations. Based on these findings it has been proposed that S. agalactiae became a pathogen through successive acquisition of exogenous virulence factors (Glaser et al., 2002). Several of these islands encode the genes of not yet characterized putative surface proteins and virulence factors. Future analysis and characterization of these genes may significantly contribute to our understanding of how pathogens evolve.
Acknowledgements The work of Gerd Bro¨ker and Barbara Spellerberg was supported by DFG grant Sp511/5-1.
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