Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes

We developed a multiplex PCR-based reverse line blot hybridizationassay (mPCR/RLB) to detect the genes encoding members of thefamily of variable surface-localized proteins of Streptococcusagalactiae (group B streptococcus [GBS]), namely, Bca (C ${alpha}$ ), Rib,Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulinA binding protein, Bac (Cß). We used the assay toidentify these genes in a collection of well-characterized GBSisolates and reference strains. The results showed that mPCR/RLBavoids the common problems of cross-reaction and nontypabilityassociated with protein typing using antisera. It is as sensitiveas, but more practical than, separate gene-specific PCRs andwould be suitable for large molecular epidemiological studiesof GBS.

INTRODUCTION

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Introduction

Group B streptococcus (GBS) (Streptococcus agalactiae) is animportant cause of sepsis, especially in neonates (19). In additionto capsular polysaccharide antigens, some S. agalactiae surfaceproteins are potential components of a protein-based GBS vaccine(16), and the use of some as carriers in polysaccharide conjugatevaccines is under investigation (5, 17). Identification of surface-localizedprotein antigens can facilitate studies of the epidemiologyand pathogenesis of GBS infection (14).

The genes encoding the proteins C ${alpha}$ (bca), Alp2 and Alp3 (C ${alpha}$ -like2 and 3) (alp2 and alp3), Epsilon (epsilon [alp1/alp5]), andRib (rib) have been well studied, and their gene sequences arepublished in GenBank (2, 16, 18, 23). They belong to a familyof surface-localized proteins containing internal tandem repeats,variation in the numbers of which causes variation in proteinsize and antigenicity (16, 23). The gene encoding the immunoglobulinA (IgA) binding protein, Cß (bac), also has been welldescribed (6, 7). In a previous study, we developed individualgene-specific PCRs for bca, rib, alp2, alp3, and bac (12), andCreti et al. have recently described a multiplex PCR methodfor bca, rib, alp2/alp3, alp4, and epsilon (2). In this study,we developed a multiplex PCR (mPCR)-based reverse line blot(RLB) hybridization assay and used it to identify seven proteinantigen genes in several sets of well-characterized GBS isolates.

MATERIALS AND METHODS

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Materials and Methods

GBS isolates and serotyping. The GBS isolates used in this study were the following: 27 referencestrains used in our previous studies (Table 1); 83 isolatesprovided by Nicola Jones, Oxford, United Kingdom, which hadbeen characterized by multilocus sequence typing (MLST) (8);58 isolates provided by Dele Davies and Shannon Manning, MichiganState University, East Lansing, MI, which also had been characterizedby MLST (3); and 50 isolates provided by Catherine Lachenauer,Channing Laboratory, that previously had been used in a serologicalstudy of protein surface antigens (15).

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TABLE 1. Conventional serotyping, molecular serotype identification based on PCR/sequencing, and mPCR/RLB hybridization protein gene profile for 27 GBS reference strains^a

Results of capsular polysaccharide serotyping were providedby the donor laboratories (1, 13).

DNA preparations. DNA was prepared from GBS cultures as described previously (13).

Molecular serotype/serosubtype and protein gene identification. Capsular polysaccharide (cps) molecular serotype/serosubtypeidentification, based on PCR/sequencing of variable regionsof the cps gene cluster, and protein gene profile, based onsingle target protein gene PCR, were performed on all isolatesused in this study, as described previously (1, 11, 13).

Oligonucleotide design. We designed two specific PCR primers and two specific probesfor each of the seven GBS protein genes, using published GenBanksequences. We also designed one GBS-specific primer pair andtwo GBS-specific probes (9) (Table 2).

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TABLE 2. Oligonucleotide primers used in this study

Primer design. The primers were designed to have similar physical characteristicsin order to allow simultaneous amplification in a multiplexreaction. Their lengths were between 20 and 32 bp, melting temperaturesbetween 57.2°C and 65.8°C, and amplicon sizes in therange of 193 to 624 bp (Table 2). The specificities of primersequences were determined by comparison with GenBank, usingFASTA, to ensure that there would be minimal risk of cross-hybridizationwith other regions of the GBS genome or unrelated genes. Allmultiplex PCR primers were 5' biotinylated to allow detectionof hybridization with a streptavidin peroxidase substrate.

Probe design. To allow optimal hybridization under the common conditions,probes were also designed to have similar physical characteristics:lengths between 20 and 24 bp and melting temperatures between57.1°C and 63.7°C (Table 2). FASTA searches were performedto compare their sequences with those in GenBank. All probeshave a 5' amine group to facilitate covalent linkage to thenylon membrane and allow membranes to be stripped and reusedrepeatedly.

Multiplex PCR system. The 25-µl eight-primer-pair mPCR mixture was preparedas follows: 5 µl template DNA, 0.25 µl each of allforward (50 pmol/µl) and reverse (50 pmol/µl) primers,1.25 µl deoxynucleoside triphosphates (2.5 mM of eachdeoxynucleoside triphosphate), 2.5 µl 10x PCR buffer,3 µl 25 mM (4.5 mM final) MgCl₂, 0.1 µl QIAGEN hotstartTaq polymerase (5 units/µl); also, water was added to25 µl. The PCR program was performed according to theQIAGEN Hotstart Taq polymerase kit instructions: 95°C for15 min, 1 cycle, 94°C for 30 s, 60°C for 30 s, 72°Cfor 1 min, 35 cycles, 72°C for 10 min, 1 cycle, and 22°Chold. A total of 8 µl of each PCR product was separatedby electrophoresis on a 1.5% agarose gel to confirm successfulamplification (13). The remaining PCR products were used forRLB hybridization (5 µl of PCR product for each).

RLB hybridization. The RLB hybridization assay was based on the method describedpreviously (21, 22), except that the hybridization temperaturewas 60°C, the conjugate used was streptavidin-peroxidase(Roche Diagnostics Co.) diluted 1:4,000 in 2x SSPE (SSPE is0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA [pH 7.7])-0.5% sodiumdodecyl sulfate, and the time of exposure to X-ray film (Hyperfilm;Amersham) was 7 min. RLB results were regarded as positive whenboth probes for a particular gene gave positive results. Tooptimize hybridization conditions, the probes were tested atseveral twofold dilutions, starting at a concentration of 0.6pM, and the final labeling concentration was generally 0.2 to0.4 pM. In our laboratory, membranes labeled with probes havebeen stripped and reused at least 20 times without any significantloss of signal (data not shown).

The sensitivity and specificity of mPCR/RLB were compared withthose of single-protein-gene-specific PCR (1, 2, 12).

Database similarity search and sequence comparison. Databases were searched for sequence similarity using the FASTAprogram in the SeqSearch program group. Sequences were comparedusing the BESTFIT and GAP programs in the Comparison programgroup. All programs are provided in WebANGIS, Australian NationalGenomic Information Service, third version.

RESULTS

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Results

The RLB results for the 27 reference strains are shown in Fig.1. Multiplex PCR products from all isolates hybridized withthe control GBS-specific probes and, with one exception, withone or more sets of protein gene-specific probes. The exceptionwas strain no. 18, which failed to hybridize with any proteingene probes on the membrane shown in Fig. 1. However, on repeattesting, it hybridized with both alp3 probes. Strain no. 24was the only exception to the rule that each strain hybridizedwith only one of the six sets of probes representing the variablesurface-localized protein family (represented by C ${alpha}$ , R, and Alp,etc.); it hybridized with both bca (C ${alpha}$ ) and rib (Rib). All eightisolates that hybridized with C ${alpha}$ gene (bca) probes also hybridizedwith Cß gene (bac) probes.

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FIG. 1. Results of mPCR/RLB hybridization for 27 reference strains. Lanes 1 to 27 represent the 27 reference strains, in the same order as in Table 1; the 20 labeled probes are in the same order as in Table 2. Duplicate rows for the same probe represent different probe concentrations (0.2 or 0.4 pM, respectively). Probe targets in rows from top to bottom are as follows: rows 1 and 2, GBS species specific; 3 and 4, blank; 5 to 8, C ${alpha}$ or Bca protein gene (protein gene profile code A [see Table 1, footnote a]); 9 to 12, blank; 13 to 16, alp2; 17 to 20, alp3; 21 to 24, blank; 25 to 28, alp4; 29 to 31, rib (protein gene profile code R); 32 to 35, epsilon/alp1/alp5; 36 and 37, Cß or Bac protein gene (protein gene profile code B); 38 and 39, blank; 40 and 41, GBS species-specific gene. PCR product of strain no. 18 hybridized with GBS-specific probes but with none of the protein gene probes. However, on retesting, its PCR product hybridized with alp3 probes.

Comparison of mPCR/RLB and single-gene-specific PCR. To confirm the sensitivity and specificity, we compared theresults of mPCR/RLB with those of single-gene-specific PCR.For all 27 reference strains (Table 1) and 191 isolates studied(Table 3), the results of mPCR/RLB were the same as those ofsingle-gene-specific PCR.

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TABLE 3. The relationship between protein gene profiles and capsular polysaccharide synthesis gene MS for 191 GBS isolates

Comparison of mPCR/RLB with conventional protein serotyping. Of the 50 isolates for which protein subtyping had been donepreviously, using antisera, 48 had reacted with two or moreantisera (15), indicating a high degree of cross-reactivity.By contrast, mPCR/RLB showed complete specificity, with no cross-reactionsbetween different gene probes (Table 3). However, one isolate(no. 24) contained two different protein genes (in additionto bac).

DISCUSSION

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Discussion

In this study, we developed an mPCR/RLB method to identify 6GBS surface protein genes and the IgA binding protein, Cß,and used it to further characterize 27 reference strains and191 isolates. With the exception of a single reference strain(no. 24 [70339] [NCTC 12907]), which contained both bca andrib, all isolates contained only one of the six members—rib,bca, epsilon/alp1/alp5, alp2, alp3, or alp4—of the variablesurface protein gene family. The Cß gene (bac) waspresent with one or other of these genes (usually bca) in 8of 27 reference strains and 16 of 191 isolates (most of whichbelonged to molecular serotype [MS] Ib).

Our study confirmed previously reported relationships betweencps serotypes and surface protein gene profiles (10, 12, 16).For example, most serotype III isolates (our molecular serosubtypes[msst] III-1 and III-2) were associated with rib (20), and msstIII-3 with alp2 (16). Serotype Ib was associated with bca andbac (14), and serotypes V and VIII with alp3 (16). However,since the relationship was not absolute, different combinationsof cps serotype and protein gene profile identified many serovariants,which will be useful in epidemiological studies and in formulationof conjugate vaccines (14, 16).

Most of the 50 isolates which had been subtyped using antiseraagainst surface proteins express "laddering" patterns in Westernblots with two or more antisera (15). This serological cross-reactivityprobably arises from conserved epitopes present within morethan one surface protein. However, in our study, all strainstested possessed one and, with a single exception, only oneof the six members of the surface protein antigen gene family—rib,bca, epsilon, alp2, alp3, and alp4 (14, 16). These results indicatethat immunologic typing of GBS surface proteins with currentlyavailable antisera is potentially misleading and should be usedwith caution, especially for epidemiological studies.

Unlike the use of antisera, molecular methods of protein "typing"do not demonstrate protein expression, which is clearly importantfor virulence. However, since the results of existing immunologicaltyping methods are so difficult to interpret, further investigationis needed to identify reliable markers of gene expression. OurmPCR/RLB assay is an appropriate method for epidemiologicaltyping, especially when combined with other molecular markers,as in our three-component system (10). If combined with an immunologicalassay, such as a Western blot, the mPCR/RLB would clarify theinterpretation of results.

The use of a polyvalent GBS conjugate vaccine, containing allof the prevalent capsular polysaccharides, has been proposedas a strategy for the prevention of GBS infections. One or moreof the GBS surface proteins could be conjugated with GBS polysaccharidesand independently contribute to protective immunity againstthe corresponding protein-containing serotypes in an effectivevaccine. In this context, a rapid, reliable molecular test capableof identifying the GBS surface protein genes, such as mPCR/RLB,would facilitate cost-effective epidemiological studies of circulatingGBS strains to identify protein genes profiles.

This mPCR/RLB method has a number of advantages over our previousprotein gene profiling method (12). It is easy to perform anddoes not require the use of gels; amplification products of43 isolates can be tested simultaneously, and therefore, themethod would be suitable for high-throughput GBS typing (4).Furthermore, it can detect all seven protein genes in a singlePCR and on one membrane, which can be reused more than 20 times.The assay (for 43 isolates) can be completed within one workingday. Although we have not tested its sensitivity compared withculture, it has the potential to be used directly for clinicalspecimens (4).

Our method also has practical advantages over the multiplexPCR described by Creti et al. (2). First, it does not involvethe use of gels, and the use of two probes to identify PCR productsincreases the sensitivity (by amplifying the signal) and specificity(the result is considered positive only if both probes are hybridized).Second, unlike Creti et al., we can distinguish alp2 from theclosely related alp3 gene by RLB. This is important, becausethese proteins classically cross-react with typing antiserabut are clearly associated with different capsular polysaccharideserotypes (alp2 with msst III-3 [3, 10] and alp3 with serotypesV and VIII [16]). Third, our mPCR/RLB method also includes theCß (IgA binding) protein gene, which is an importantvirulence factor.

Finally, because this method uses only 20 of 43 lanes in theMiniBlotter, additional targets can be added to both mPCR andRLB, such as serotype-specific cps sequences (11) or mobilegenetic elements, which are included in our three-componentgenotyping system (10), or antibiotic resistance genes.

In conclusion, our mPCR/RLB assay is a more sensitive and specificmethod for the identification of GBS surface protein gene profilesthan the use of antisera and more efficient and convenient thanseparate PCRs or multiplex PCR with gel electrophoresis. Applicationof PCR/RLB in future epidemiological studies and for surveillancewill facilitate the appropriate formulation of candidate GBSvaccines.

ACKNOWLEDGMENTS

We thank our colleagues for providing GBS isolates other thanthose isolated in our laboratory. Lawrence Paoletti and CatherineLachenauer, Channing Laboratory, Boston, Mass., provided 11reference strains, including nine strains of GBS serotypes (Iato VIII) and another two reference strains (BM110 and A909);Diana Martin, Streptococcus Reference Laboratory, at the Instituteof Environmental Science and Research Limited, Wellington, NewZealand, provided nine international reference GBS type strains,including strains of serotypes Ia to VI; Johan Maeland, Departmentof Microbiology, School of Medicine, Norwegian University ofScience and Technology, Trondheim, Norway, provided seven serotypereference strains used in his previous studies; Nicola Jones,Nuffield Department of Clinical Laboratory Sciences, Institutefor Molecular Medicine, John Radcliffe Hospital, Oxford OX39DU, United Kingdom, provided 83 GBS strains used in a previousMLST study; 58 isolates were provided by Dele Davies and ShannonManning, Michigan State University, East Lansing, Mich., whichhad been used previously in a study of MLST; and 50 clinicalisolates were from Catherine Lachenauer, Channing Laboratoryand Division of Infectious Diseases, Department of Medicine,Brigham and Women's Hospital, Children's Hospital, Boston, MA02115.

FOOTNOTES

* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyng{at}icpmr.wsahs.nsw.gov.au.

REFERENCES

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