Journal of Bacteriology, March 2004, p . 1398-1408, Vol . 186, No . 5

Relevance of Peptide Uptake Systems to the Physiology and Virulence of Streptococcus agalactiae

Ulrike Samen,¹ Birgit Gottschalk,¹ Bernhard J . Eikmanns,¹ and Dieter J . Reinscheid^1*

Department of Microbiology and Biotechnology, University of Ulm, D-89069 Ulm, Germany¹

Received 12 September 2003/ Accepted 21 November 2003

 
Streptococcus agalactiae is a major cause of invasive infections in human newborns . To satisfy its growth requirements, S . agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S . agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits . Oligopeptide uptake by S . agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins [OppA1 and OppA2] with overlapping substrate specificities . Dipeptides were found to be taken upin parallel by the oligopeptide permease OppA1-F, by the dipeptideABC transporter DppA-E, and by the dipeptide symporter DpsA.Reverse transcription-PCR analysis revealed a polycistronicorganization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S . agalactiae . The results of quantitativereal-time PCR revealed a medium-dependent expression of theoperons dppA-E and oppA1-F in S . agalactiae . Growth of S . agalactiaein human amniotic fluid was shown to require an intact dpsAgene, indicating an important role of DpsA during the infectionof the amniotic cavity by S . agalactiae . Deletion of the oppBgene reduced the adherence of S . agalactiae to epithelial cellsby 26%, impaired its adherence to fibrinogen and fibronectinby 42 and 33%, respectively, and caused a 35% reduction in expressionof the fbsA gene, which encodes a fibrinogen-binding proteinin S . agalactiae . These data indicate that the oligopeptidepermease is involved in modulating virulence traits and virulencegene expression in S . agalactiae.

 
Streptococcus agalactiae has a limited capacity to synthesize amino acids and must acquire from the environment 9 of the 20 proteinogenic amino acids for growth [38] . The amino acid requirementsof S . agalactiae can be satisfied by the uptake of peptides,which are cleaved in the cytoplasm to their respective aminoacids [69, 70] . In microorganisms, two different peptide uptakesystems have been described [23, 41] . The most common peptidetransporters are binding-protein-dependent permeases, consistingof multiple components, belonging to the ATP-binding cassette[ABC] transporter superfamily [24] . The process of peptide transportby ABC transporters involves the extracytoplasmic binding ofthe substrate by a substrate-binding protein, transfer of thesubstrate to two membrane-integrated permeases for translocationacross the cytoplasmic membrane, and ATP hydrolysis by one ortwo proteins located on the cytoplasmic side of the membrane[24] . In numerous bacteria, the uptake of di- and oligopeptidesis mediated by these ABC transporters [41] . In contrast, somemicroorganisms take up di- and/or tripeptides by single proteinswith similarity to eukaryotic peptide transport proteins ofthe PRT family [62] . These bacterial permeases use the protonmotive force across the cytoplasmic membrane for the uptakeof peptides [23, 37] . They exhibit a broad substrate specificity, but size recognition is restricted to di- and tripeptides only[16] . Examples of this kind of peptide symporter are the tripeptide permeases from Escherichia coli and Salmonella enterica serovar Typhimurium [17, 60], and the di- and tripeptide permeases [DtpT]of Lactococcus lactis and Lactobacillus helveticus [23, 37].

In L . lactis, whose peptide uptake systems have been the focus of intense research, di- and oligopeptides are taken up by two distinct ABC transporters, termed DppA-E and OppA-F, respectively[52, 64] . Di- and tripeptide uptake is also mediated in L . lactisby the proton-driven permease DtpT [23] . In contrast, in Streptococcuspyogenes, dipeptides are taken up exclusively by the ABC transportersystem DppA-E [44].

In addition to nutrient uptake, peptide permeases of several organisms are involved in sensing pertinent environmental signalsin the form of peptides, culminating in diverse responses suchas sporulation [51], chemotaxis [1, 32], conjugation [29], development of competence [4, 5, 26], or expression of virulence determinants[11, 34, 44, 45] . Signature-tagged mutagenesis identified theputative dipeptide ABC transporter DppA-E as a requirement forgrowth and survival of S . agalactiae in a rat sepsis model [28], suggesting an important role of peptides as nutrients or signaling molecules for the virulence of these bacteria.

S . agalactiae is the predominant cause of septicemia, pneumonia, and meningitis in neonates and poses a significant threat to parturient women [8, 54] . A common cause of neonatal infectionis the colonization of the human rectovaginal tract with S.agalactiae, ascending spread of the bacteria into the amnioticfluid, and aspiration of contaminated amniotic fluid by theinfant [8] . Although amniotic fluid contains only low amountsof free amino acids [35], it supports growth of S . agalactiaeto high concentrations [14, 66], indicating that S . agalactiae satisfies its amino acid requirements in amniotic fluid by theuptake of peptides.

Recently, the genome sequences of the two S . agalactiae strains NEM316 and 2603 V/R were published, thereby allowing a genome-wide search for putative peptide permease genes in S . agalactiae.

The present report describes the characterization of four putative peptide permeases in S . agalactiae . Defined mutants in the putative peptide uptake systems were constructed and used to define the role of these permeases in the uptake of di- and oligopeptidesand in the growth of S . agalactiae in various complex media.The transcriptional organization of the peptide permease geneswas analyzed in S . agalactiae, and additionally, the expressionof these genes was determined during growth in various media.Finally, we addressed the importance of the peptide permeasesin the binding of S . agalactiae to host proteins, in the attachmentto eukaryotic cells, and in the expression of a virulence genein S . agalactiae.

 
Bacterial strains, eukaryotic cells, and growth conditions. The S . agalactiae strain O90R [ATCC 12386] is a capsule mutantof the serotype Ia clinical isolate O90 . Strain O90R was usedfor the construction of insertion and deletion mutants in genesencoding putative peptide transporters in S . agalactiae . S.agalactiae strains belonging to the serotypes Ia, Ib, II, III,IV, and V, respectively, are clinical isolates that were describedpreviously [53]. S . agalactiae was generally grown in Todd-Hewittbroth supplemented with 1% yeast extract [THY] . RecombinantS . agalactiae strains were selected in the presence of erythromycin [5 µg/ml] . Growth experiments with S . agalactiae were performed in THY, in fetal calf serum [FCS], and in amnioticfluid . Amniotic fluid was obtained by amniocentesis from a patientsuffering from polyhydramnios, and was kindly provided by thechildren's hospital in Ulm, Germany . Growth of S . agalactiaein THY or FCS was monitored by determining the optical densityof the culture at 600 nm [OD₆₀₀] . Due to the turbid nature ofamniotic fluid, growth of S . agalactiae in this medium was determined by measuring the viable cell counts of the culture . In additionto growth in complex media, S . agalactiae was also grown in chemically defined medium [CDM] [65] . Depleted CDM [dCDM] was prepared without the essential amino acid isoleucine but containing isoleucine-containing di-, tri-, and oligopeptides at a final concentration of 0.1 mg ml^-1 . The isoleucine-carrying peptides were purchased from Bachem [Bubendorf, Switzerland] . The amino acid requirements of different S . agalactiae strains were tested with dCDM lacking, in each case, 1 of the 20 proteinogenic amino acids . Amino acids in the growth medium were detected as their o-phthaldialdehyde derivatives via high-pressure liquid chromatography as described elsewhere [68].

E . coli DH5 was grown in Luria-Bertani medium, and pG⁺host6-or pTCV-lac-carrying clones were selected with ampicillin [100µg/ml] or kanamycin [50 µg/ml].

The cell line A549 [ATCC CCL-185] was obtained from the American Type Culture Collection . A549 is a human lung carcinoma cellline which has many characteristics of type I alveolar pneumocytes.A549 cells were propagated in RPMI tissue culture medium [GibcoBRL] supplemented with 10% FCS . Tissue cultures were incubatedin a humid atmosphere at 37°C with 5% CO₂.

Construction of insertion and deletion mutants in S . agalactiae. Primers used for the construction of insertion or deletion mutantsof S . agalactiae O90R are listed in Table 1 . Insertional inactivationof genes in S . agalactiae O90R was performed as described previously[48] . Briefly, an internal fragment of the target gene was amplifiedby PCR and cloned into the thermosensitive plasmid pG⁺host6.The resulting plasmid was transformed into S . agalactiae accordingto the method described by Ricci et al . [49], and transformants were selected on erythromycin agar at 30°C . Cells carryingthe plasmid within the chromosome were selected at 37°Cunder erythromycin pressure as described previously [31] . The locations of the plasmid insertions were confirmed by Southern blot hybridization . The disruption of the oppA2 gene in S . agalactiae was confirmed by ClaI digestion of genomic DNA of the parental strains and of putative oppA2 integration mutants . Insertional inactivation of the gbs1577 gene was tested with HpaI-digested chromosomal DNA of the parental strains and of putative gbs1577 integration mutants . Digoxigenin-labeled probes of the genes oppA2 and gbs1577 were obtained with the primers 5'-GGATTGGAATGGTTCAAATGGand 5'-AGAGTCCGAATCAGCTGTG and with the oligonucleotides 5'-TTGAAGAGTCTAAAGGTGGand 5'-ACTTATTCCCTGCGAACTC, respectively . Integration mutantswere constructed in the S . agalactiae O90R wild type and inS . agalactiae mutants that already carried deletions in thegenes oppA1, oppB, dppB, and/or dpsA . The genes dppA, dppB, oppA1, oppB, and/or dpsA were deleted from S . agalactiae accordingto a procedure described by Schubert et al . [53] . In brief,two DNA fragments flanking the target gene were amplified byPCR with the primers listed in Table 1 . Due to 21 bases of cDNAin the two primer sets used for the amplification, the resultingPCR products possessed a stretch of 21 identical base pairs.This sequence identity was subsequently used to combine the two PCR products in a crossover PCR . The resultant PCR product consisted of a single DNA fragment that carried the flankingregions of the target gene . The crossover PCR product was clonedinto the plasmid pG⁺host6, and the construct was used to transform S . agalactiae with subsequent erythromycin selection at 30°C. Cells in which the plasmid had integrated into the chromosomewere selected at 37°C under erythromycin pressure . Six suchintegrant strains were serially passaged for 6 days in liquidmedium at 30°C without erythromycin selection to facilitatethe excision of the pG⁺host6 construct, leaving the desiredtarget gene deletion in the chromosome . Dilutions of the seriallypassaged cultures were plated onto agar, and single colonieswere tested for erythromycin sensitivity to identify pG⁺host6excisants . Successful deletion of the genes oppA1, oppB, dppA,and/or dppB was confirmed by Southern blotting with EcoRI-digested chromosomal DNA of the S . agalactiae parental strains and of the putative deletion mutants . A digoxigenin-labeled probe for the detection of deletions in oppA1 and/or oppB was obtained by PCR with the primers 5'-GTATCTTGATAACAGACG and 5'-GAATACCTACGATGATGCGC. Deletions in the genes dppA and/or dppB were detected with a digoxigenin-labeled probe that had been obtained by PCR with the primers 5'-GTGATACACCGCTACTTC and 5'-CCTTGAGCACCTTTCCCAAC.The successful deletion of the dpsA gene was confirmed by Southern blot analysis with XbaI-digested chromosomal DNA from S . agalactiae and a digoxigenin-labeled probe obtained by PCR with the primers 5'-AACTTTTTGACTGCGTGAC and 5'-TAGGTGCTTTGACCGAGATG . Deletion mutants that had been confirmed by Southern blot analysis were subsequently used for the construction of double and triplemutants in genes encoding other putative peptide transporters.

 
RNA preparation, RT-PCR, and LightCycler real-time PCR. S . agalactiae O90R was grown to mid-log phase in 50 ml of THY,CDM, dCDM plus dipeptide [Ile-Val], or dCDM plus oligopeptide [Val-Tyr-Ile-His-Pro-Phe] . Total RNA was prepared from the bacterial culture with the RNeasy midi extraction kit [Qiagen] and treatedfor 1 h with 150 U of RNase-free DNase [Promega] . RNA sampleswere checked for DNA contamination by PCR without prior reverse transcription [RT] . As no amplicons were obtained, DNA contamination during RNA preparation could be excluded . The transcriptional organization of the genes dppA-E, oppA1-F, and dpsA in S . agalactiaewas elucidated by RT-PCR analysis with the primers listed inTable 2 by using 1-µg RNA samples prepared from THY-grownculture.

 
Quantitative real-time PCR experiments were performed afterRT of RNA with random hexanucleotides and the RevertAid first-strandcDNA synthesis kit [MBI Fermentas] according to the instructionsof the manufacturer . Expression analysis of the genes dppA,dppB, dppE, oppA1, oppB, oppF, and dpsA, respectively, was performedwith the primers listed in Table 2 in a LightCycler as describedpreviously [21] . The quantity of cDNA for the investigated geneswas normalized to the quantity of gyrA cDNA in each sample.Each experiment was performed at least four times with two independentRNA preparations.

Construction of the fbsA-lacZ transcriptional fusion plasmid pTfbsA. Plasmid pTCV-lac [46] was used for expression analysis of thefbsA gene in S . agalactiae . A DNA fragment of 747 bp containingthe fbsA promoter [unpublished data] was amplified by PCR fromthe genome of S . agalactiae with the primers 5'-CCGGAATTCAACTATTGCTCCCCTGC and 5'-GCCGGATCCTTTCCTGATTTCCAAGTTC . The EcoRI and BamHI sitesused for cloning are underlined . After digestion of the PCR product and of plasmid pTCV-lac with EcoRI and BamHI, the fbsApromoter region was ligated into pTCV-lac, resulting in plasmidpTfbsA . The plasmids pTCV-lac and pTfbsA were subsequently transformedinto S . agalactiae by electroporation [49], and recombinantclones were obtained by erythromycin selection.

Binding of fluorescein isothiocyanate-labeled group B streptococci to immobilized human proteins. Terasaki microtiter plates were coated with the human proteinsfibrinogen, fibronectin, and haptoglobin, respectively, andthe binding of fluorescein isothiocyanate-labeled S . agalactiaeto the immobilized fibrinogen was measured as described previously[21] . Results were measured in triplicate, and each assay wasrepeated at least four times.

Adherence and internalization assays. Adherence of S . agalactiae to A549 epithelial cells and internalizationinto this cell line were assayed as described previously [21]. Briefly, A549 cells were transferred to 24-well tissue culture plates at approximately 4 x 10⁵ cells per well and cultivatedovernight in RPMI tissue culture medium supplemented with 10%FCS . After replacement of the medium with 1 ml of fresh medium,the cells were infected with S . agalactiae at a multiplicityof infection of 10:1 and incubated at 37°C for 2 h . Theinfected cells were subsequently washed three times with phosphate-bufferedsaline . The number of cell-adherent bacteria was determinedby lysis of the eukaryotic cells with distilled water and subsequentdetermination of CFU by plating appropriate dilutions of thelysates on THY agar . Intracellular bacteria were determined after a further incubation of the infected cells for 2 h with RPMI medium containing penicillin G [10 U] and streptomycin[0.01 mg] to kill extracellular bacteria . After three washeswith phosphate-buffered saline, the epithelial cells were lysedwith distilled water and the amount of intracellular bacteriawas quantitated by plating serial dilutions of the lysate ontoTHY agar plates . All samples were tested in triplicate, andexperiments were repeated at least three times.

Determination of ß-galactosidase activity. Recombinant S . agalactiae strains harboring the plasmid pTCV-lacor pTfbsA were grown aerobically in THY broth at 37°C withshaking [100 rpm] . At different time points, 1-ml samples werewithdrawn to determine the OD₆₀₀ and ß-galactosidaseactivity of the culture . Prior to measuring ß-galactosidaseactivity [36], cells were permeabilized with a 0.5% toluene-4.5%ethanol mixture as described previously [46] . Enzymatic activityis expressed in Miller units, which were calculated as follows:[10³ x OD₄₂₀]/[time of the reaction [in minutes] x OD₆₀₀ x dilutionof the cells in the assay].

Nucleotide sequence accession number. The genomic DNA sequence of S . agalactiae NEM316 can be accessedin the EMBL database under accession number AL732656 . In theSwissProt TREMBL database, the polypeptides described in thisreport possess the following accession numbers: DppA, Q8E7H0; DppB, Q8E7G9; DppC, Q8E7G8; DppD, Q8E7G7; DppE, Q8G7G6; OppA1, Q8E7L0; OppA2, Q8E5L7; OppB, Q8E7K9; OppC, Q8E7K8; OppD, Q8E7K7; OppF, Q8E7K6; DpsA, Q8E489.

 
The genome of S . agalactiae possesses several putative peptide permease genes. The annotated genome sequence of the S . agalactiae strains NEM316[http://genolist.pasteur.fr/SagaList/index.html] and 2603 V/R [http://www.pnas.org/cgi/data/182380799/DC1/1] indicated the presence of three ABC transporters and one proton-driven symporter that are possibly involved in the import of peptides into the cell . In the genome of S . agalactiae NEM316, the putative ABC peptide transporters are encoded by the genes gbs0144 to gbs0148, gbs0184 to gbs0188, and gbs1573 to gbs1577 . Accordingto the functional analysis of these genes [see below], the S.agalactiae genes gbs0144 to gbs0148 were termed oppA1-F andthe genes gbs0184 to gbs0188 were named dppA-E [Fig . 1] . As the S . agalactiae genome carries two oppA homologs [63], onecarried by gbs0144 and the other carried by gbs0966, the twogenes were termed oppA1 and oppA2, respectively . The putativefunction of each of the gene products of the ABC transporterswas inferred based on homology to functionally characterizedABC transporters [24] . Thus, the proteins DppA, OppA1, OppA2,and Gbs1577 represent putative substrate-binding proteins, thepolypeptides DppB,C, OppB,C, and Gbs1575,1576 represent hydrophobicintegral membrane proteins, and DppD,E, OppD,F, and Gbs1573,1574represent putative ATPases that couple ATP hydrolysis to substratetransport across the membrane.

 
The genome of S . agalactiae also contains a gene, gbs1513, whose derived polypeptide sequence reveals 49% identity to the di- and tripeptide permease DtpT from L . lactis [23] . Functionalanalysis of gbs1513 in S . agalactiae indicated that it encodesa dipeptide symporter [see below], and therefore, gbs1513 wastermed dpsA.

Amino acid auxotrophies of S . agalactiae isolates. Studying peptide uptake in S . agalactiae requires knowledge regarding amino acid auxotrophies of the strains in use . Ina 1943 report, S . agalactiae strains were shown to be auxotrophicfor the amino acids valine, leucine, isoleucine, phenylalanine,glutamic acid, arginine, lysine, histidine, and tryptophan [38]. We tested 12 different S . agalactiae strains, belonging to six different serotypes, for growth in CDM lacking, in each case, one of the 20 proteinogenic amino acids . All of the S . agalactiae isolates investigated, including strain O90R, were auxotrophic for the amino acids valine, leucine, isoleucine, phenylalanine, tyrosine, arginine, lysine, histidine, and tryptophan . The tested S . agalactiae isolates therefore differ from the previously described strains [38] in that they require tyrosine but not glutamic acid for growth . Analysis of metabolic pathways that can be predicted from the genome sequence [http://www.genome.ad.jp/kegg/metabolism.html] of the S . agalactiae strains NEM316 and 2603 R/V confirmed for these strains the amino auxotrophies identified by the growth experiments . Peptide uptake was further investigated in S . agalactiae strain O90R by making use of its isoleucine auxotrophy.

Identification of the oligopeptide uptake system in S . agalactiae. Oligopeptide import in S . agalactiae was studied by deleting the genes oppA1 and oppB and by insertionally inactivating the oppA2 gene in the genome of S . agalactiae O90R, resulting in the mutant strains oppA1 oppB and oppA2-int, respectively . Furthermore,a double mutant of the genes oppA1 and oppA2 was constructedand named oppA1 oppA2-int . In THY complex medium, S . agalactiaeO90R and its isogenic mutants revealed identical growth ratesand final ODs [data not shown], suggesting that the genes oppA1,oppA2, and oppB are dispensable for growth in this medium . Therole of these genes for the uptake of peptides was studied bygrowing strain O90R and its isogenic mutants in various formulationsof CDM . All strains grew in complete CDM [Fig . 2] and, as determined by monitoring the OD of the cultures during growth, showed approximately identical growth rates and final ODs [data not shown] . WhenCDM was depleted of the essential amino acid isoleucine [dCDM],there was no apparent growth of the strains [Fig . 2], confirming the isoleucine auxotrophy of S . agalactiae O90R . Addition of the isoleucine-containing dipeptide Ile-Val to dCDM restored growth of all the strains tested [Fig . 2], suggesting that the isoleucine auxotrophy can be supplemented by an isoleucine-containing dipeptide . This finding also indicates that the genes oppA1, oppA2, and oppB are not essential for the uptake of dipeptides.Also demonstrated in Fig . 2, S . agalactiae O90R revealed growthin dCDM with isoleucine-containing tri-, tetra-, and hexapeptides.However, no growth of the mutant oppB was observed in thesemedia [Fig . 2 and Table 3], indicating an essential functionof oppB for the uptake of oligopeptides by S . agalactiae . Interestingly, the mutant strain oppA1 and oppA2-int exhibited growth in dCDMwith different tri-, tetra-, and hexapeptides while the doublemutant oppA1 oppA2-int did not grow on isoleucine-containingoligopeptides [Fig . 2] . These data suggest that the substrate-binding proteins OppA1 and OppA2 are both involved in oligopeptide uptake and that they can substitute for each other functionally.

 
Identification of dipeptide import systems in S . agalactiae. Dipeptide uptake was studied by deleting the genes dppA, dppB, and dpsA and insertionally disrupting the gene gbs1577 in thechromosome of S . agalactiae O90R, resulting in the mutant strains dppA, dppB, dpsA, and gbs1577-int . In THY complex medium, themutants revealed no difference in growth rate and final OD comparedto S . agalactiae O90R [data not shown] . All of the mutants,however, exhibited growth in dCDM that contained isoleucine-carrying dipeptides [Table 3], indicating that the import of dipeptides in S . agalactiae is mediated by different uptake systems with overlapping substrate specificities . This hypothesis was tested by constructing S . agalactiae double and triple mutants with different combinations of defects in the genes dppB, oppB, gbs1577,and dpsA . All double mutants revealed growth in dCDM with isoleucine-containingdipeptides [Table 3], suggesting that more than two uptake systemsare involved in the import of dipeptides in S . agalactiae . Finally,a triple mutant with deletions in the genes dppB, oppB, and dpsA did not grow in dCDM with the isoleucine-carrying dipeptides Ile-Val, Ile-Glu, and Ile-Pro, respectively . This finding showsthat dipeptide uptake is mediated by the oligopeptide permeaseOppA1-F, the dipeptide permease DppA-E, and the dipeptide symporterDpsA . Additionally, it shows that any of these permeases issufficient for growth of S . agalactiae with dipeptides as anamino acid source . Our data also indicate that the putativeABC transporter genes gbs1573 to gbs1577 are not required fordipeptide import . However, the triple mutant dppB oppB dpsA was still capable of growth on the dipeptide Ile-Phe, suggesting the presence of at least one more dipeptide uptake system in S . agalactiae . High-pressure liquid chromatography analysis did not detect free isoleucine during growth of S . agalactiae in Ile-Phe-containing dCDM [data not shown], indicating thatthis dipeptide is not externally hydrolyzed by S . agalactiae.

Transcriptional organization and expression control of dppA-E, oppA1-F, and dpsA. The transcriptional organization of the genes dppA-E, oppA1-F, and dpsA was investigated by RT-PCR with total RNA from S . agalactiaeO90R . As shown in Fig . 3A, RT-PCR amplified the regions dppAto dppC and dppB to dppE from S . agalactiae RNA, suggestingthat the genes dppA-E comprise an operon . No amplification products were obtained with primer pairs specific to the genes gbs0183 to dppA and dppD to gbs0189, indicating transcriptional initiationupstream of dppA and transcriptional termination downstreamof dppE, respectively . Similarly, RT-PCR amplified the regionsoppA1 to oppB, oppB to oppC, and oppC to oppF from O90R RNA[Fig . 3A], suggesting transcriptional coupling of the genesoppA1-F . Here again, no amplification products were obtainedwith primer pairs specific to the genes gbs0143 to oppA1 andoppD to rDNA, indicating a promoter in front of oppA1 and a transcriptional terminator downstream of oppF . Analysis of the transcriptional organization of dpsA by RT-PCR revealed a dpsA-specific amplicon with dpsA-specific primers [Fig . 3A] . No amplificationproducts were obtained by RT-PCR with primers specific to theregions gbs1512 to dpsA and dpsA to gbs1514, indicating a monocistronicorganization of the dpsA gene in S . agalactiae . All of the primerpairs used for RT-PCR analysis amplified products of the expectedsize from chromosomal S . agalactiae DNA [Fig . 3B] . As no PCRproduct was amplified from total S . agalactiae RNA [data notshown], DNA contaminations in the RNA preparation could be excluded.

 
In several organisms, transcriptional attenuation behind dppA or oppA results in an increased amount of dppA- or oppA-specific mRNA compared to the polycistronic mRNA [2, 10, 44] . Therefore, quantitative real-time PCR was performed in a LightCycler to determine the ratio of expression between dppA and the downstream genes, dppB and dppE, and between oppA1 and the downstream genes,oppB and oppF . The impact of medium components on the expressionof dppA, dppB, dppE, oppA1, oppB, oppF, and dpsA was also studied by quantitative real-time PCR . Expression analysis was performed with total RNA from S . agalactiae O90R after growth in THY, CDM, or dCDM supplemented with the dipeptide Ile-Val or the oligopeptide Val-Tyr-Ile-His-Pro-Phe . Under different growth conditions, the expression of the dppA gene was similar to that of dppE and even weaker than that of dppB [Fig. 4A] . Similarly,the amount of oppA1 mRNA was, under different growth conditions,nearly identical to the amount of transcript from the genesoppB and oppF [Fig. 4B] . These findings suggest that in theoperons dppA-E and oppA1-F in S . agalactiae, transcriptionalattenuation does not take place behind the genes dppA and oppA1.

 
As depicted in Fig . 4, the expression of the oppA1-F operonsignificantly exceeded the expression of the dppA-E operon andthat of the dpsA gene [note the different scale of the transcriptcopy number between the graphs], indicating that the oligopeptidepermease might be required in higher amounts by the cell . Growthof S . agalactiae under different culture conditions revealeda moderately increased transcription of the genes dppA, dppB,and dppE in THY complex medium . In contrast, expression of thedpsA gene remained constant under the growth conditions tested[Fig . 4A] . Interestingly, the transcription of the genes oppA1,oppB, and oppE was about fivefold induced in dCDM containingthe hexapeptide Val-Tyr-Ile-His-Pro-Phe [Fig . 4B] . However, growth of S . agalactiae in dipeptide-containing dCDM had no effect on the expression of either of the peptide permease-encoding genes . These findings indicate complex regulatory mechanisms that control the expression of peptide permease-encoding genesin S . agalactiae.

The dpsA gene plays an important role during growth in amniotic fluid. S . agalactiae is known to infect the placenta and grow in amnioticfluid to high cell densities [15, 50, 66] . The importance of peptide uptake for growth of S . agalactiae in amniotic fluid was studied with the strain O90R and the triple mutant dppB oppB dpsA . As shown in Fig . 5, the mutant dppB oppB dpsA grewto significantly lower cell numbers in amniotic fluid than S. agalactiae O90R, suggesting an important role of dipeptide and/or oligopeptide uptake during growth of S . agalactiae in amniotic fluid . To investigate this effect in more detail, growth in amniotic fluid was monitored for the S . agalactiae single mutants dppB, oppB, and dpsA [Fig . 5] . Interestingly, the mutant dpsA revealed the same growth defect in amniotic fluid as observed for the triple mutant dppB oppB dpsA, suggesting that the dpsAdeficiency caused the growth impairment in amniotic fluid . Thedifferent peptide permease mutants and strain O90R were alsotested for growth in various complex media . However, the testedstrains did not reveal differences in growth rates and finalODs in either THY or FCS [data not shown] . These findings suggestthat S . agalactiae specifically requires the dipeptide permeaseDpsA to successfully proliferate in human amniotic fluid.

 
The oligopeptide permease is important for the binding of S . agalactiae to host proteins. In Streptococcus gordonii and Streptococcus pneumoniae, therespective oligopeptide permeases control the interaction ofthe bacteria with host proteins [11, 27, 34] . Therefore, theimportance of the three peptide uptake systems for the bindingof S . agalactiae to different host proteins was investigated.S . agalactiae strain O90R revealed significant binding to immobilizedfibrinogen, fibronectin, and haptoglobin [Table 4] . However,the fibrinogen binding of the S . agalactiae mutants oppB and dppB oppB dpsA was reduced by 42% . Similarly, both mutants revealeda 33% reduction in binding to human fibronectin . None of thetested mutants was impaired in its interaction with human haptoglobin.These findings indicate that the oligopeptide permease in S.agalactiae plays a role in the binding of the bacteria to humanfibrinogen and fibronectin.

 
The influence of peptide permeases on the bacterial adherence to and invasion of eukaryotic cells. In S . pneumoniae, the oligopeptide permease was shown to berequired for efficient adherence of the bacteria to human cells[11] . The S . agalactiae wild-type strain O90R and the peptidepermease mutants dppB, oppB, dppB dpsA, and dppB oppB dpsA weretherefore tested for their ability to adhere to and invade cells of the human lung epithelial cell line A549 . Among the tested strains, the mutants oppB and dppB oppB dpsA revealed a 26%reduced adherence to host cells . However, none of the mutantsexhibited altered invasion into host cells [data not shown], indicating that a deficiency in the oligopeptide permease only moderately reduces adherence of S . agalactiae to human cells.

The oligopeptide permease controls expression of the fbsA gene. In S . agalactiae, the FbsA protein represents the major fibrinogen-bindingprotein [53], which also plays an important role in the adherenceof the bacteria to host cells [unpublished data] . As the previousresult demonstrated an influence of the oligopeptide permeaseon fibrinogen binding and adherence of S . agalactiae to humancells, the expression of the fbsA gene was analyzed in S . agalactiaeO90R and its isogenic mutant oppB . Both strains were transformedwith the vector pTCV-lac and with the plasmid pTfbsA, whichcarries the fbsA promoter in front of the promoterless lacZgene . In pTfbsA-carrying strains, fbsA expression can be monitoredby measuring the ß-galactosidase activity . Strainsthat harbored the vector pTCV-lac did not possess ß-galactosidaseactivity during growth [data not shown] . However, both pTfbsA-carryingstrains revealed a growth-phase-dependent expression of fbsA,peaking in the middle of the exponential growth phase with amoderate decline during late logarithmic and stationary growth[Fig . 6] . The obtained results are identical to the expressionanalysis of fbsA in the S . agalactiae strain 6313-fbsA-luc [21], which carries in its chromosome a promoterless luciferase gene fused to the fbsA gene . This result indicates that plasmid-mediated expression of fbsA does not influence its growth-phase-dependent regulation . Growth experiments revealed identical growth ofthe S . agalactiae strains O90R[pTfbsA] and oppB[pTfbsA] [Fig.6] . However, in strain oppB[pTfbsA], the fbsA expression wasapproximately 35% reduced compared to the wild-type strain O90R[pTfbsA],indicating that the oligopeptide permease stimulates the expressionof the fbsA gene in S . agalactiae.

 
S . agalactiae is a fastidious organism that requires 9 of the 20 proteinogenic amino acids for growth [reference 38 and the present report] . To meet its amino acid requirements, S . agalactiae takes up amino acids in their free or peptide-bound form . Peptides are imported by the cell with the aid of specific peptide permeases and are subsequently cleaved by intracellular peptidases to their single amino acids [41] . Four putative peptide permeaseswith homology to known peptide transport systems have been notedin the genomes of the two sequenced S . agalactiae strains [18,63] . However, none of these putative peptide permeases had beentested for functionality . In the present report, defined mutantsin the four putative peptide permease systems were constructedand used to define the roles of these uptake systems in theoverall process of peptide utilization and the expression ofvirulence traits in S . agalactiae.

Feeding experiments with defined S . agalactiae mutants demonstrated that peptide uptake in these bacteria is mediated by two ABC transporters and one putative proton-driven permease, as isalso the case for L . lactis and E . coli [23, 41, 52, 60, 64].The ABC transporter Gbs1573-1577, however, appears not to berequired for the uptake of peptides in S . agalactiae . As Gbs1573-1577reveals similarity to peptide transporters but also to Ni²⁺ permeases [18], it is possibly involved in the uptake of Ni²⁺.Like other microorganisms [41], S . agalactiae requires an ABC transporter system [OppA1-F] for the import of oligopeptidesof two to six residues . In the genome of S . agalactiae, two oppA homologs, termed by us oppA1 and oppA2, have been noted. The presence of two oppA genes readily explains the apparent dispensability of either for the transport of oligopeptidesby the oligopeptide permease system . Indeed, the two proteinsappear to have overlapping specificities for oligopeptides,as only the simultaneous inactivation of oppA1 and oppA2 abolished transport of oligopeptides . The presence of several oligopeptide binding proteins that serve the same membrane complex is rather common in microorganisms . S . pneumoniae, S . gordonii, and E. coli each possess three genes that encode oligopeptide binding proteins [3, 26, 40] . In Borrelia burgdorferi, five oppA homologshave been identified [9] . Finally, Enterococcus faecalis produces two peptide binding proteins with different affinities for the peptide sex pheromone cCf10, which is required for conjugative transfer of certain plasmids [29] . It has been suggested that,as OppA1 and OppA2 from S . agalactiae are closely related toeach other on the amino acid level, they presumably evolvedfrom a common ancestor following gene duplication [63].

Dipeptide import by S . agalactiae appears to be a complex process, involving the two ABC transporters DppA-E and OppA1-F and the putative proton-driven symporter DpsA . DpsA and DppA-E homologsof several microorganisms have been shown to transport bothdipeptides and tripeptides [6, 22, 37, 41, 67] . The tripeptide Val-Trp-Ile was not transported in oligopeptide permease mutantsof S . agalactiae, demonstrating that the dipeptide permeases DppA-E and DpsA are not involved in the transport of this tripeptide. However, this finding does not rule out other tripeptides as substrates for either of the two dipeptide permeases.

Unexpectedly, feeding experiments with S . agalactiae peptide permease mutants demonstrated the uptake of the dipeptide Ile-Phe even in the absence of functional DppA-E, OppA1-F, and DpsA permeases . Cleavage of the dipeptide Ile-Phe by extracellular peptidases is unlikely, as isoleucine was not detectable in Ile-Phe-containing dCDM and other isoleucine-containing dipeptides also were not cleaved by S . agalactiae . Our findings therefore indicate the presence of a further dipeptide permease that mediates the uptake of the dipeptide Ile-Phe in S . agalactiae . Interestingly, L . lactis triple mutants in the two ABC peptide transporters and the proton-driven peptide symporter also still grow on different dipeptides [52] . The presence of four different dipeptide permeasesis therefore discussed for L . lactis as well.

The presence of different dipeptide uptake systems in S . agalactiae indicates their involvement in the import of different typesof dipeptides . However, the S . agalactiae mutants dppB, oppB, and dpsA exhibited unaltered growth in dCDM with different isoleucine-containing dipeptides, suggesting overlapping substrate specificities of the three dipeptide uptake systems . In contrast, mutant dpsA was significantly impaired for growth in human amniotic fluid, whereas the mutants dppB and oppB exhibited identical growthcompared to the parental strain O90R . Amniotic fluid has a lowconcentration of free amino acids [55] but contains significantamounts of proteins and peptides [47, 56] . It is therefore temptingto speculate that during growth of S . agalactiae in human amnioticfluid, the permease DpsA plays an essential role in the importof specific dipeptides to satisfy the bacterial amino acid requirements. Similarly, L . lactis also depends on the DpsA ortholog DtpT for growth in a mixture of caseins [59] . A frequent cause of S . agalactiae infection is aspiration of infected amniotic fluid by the fetus . Amniotic fluid only has low titers of antibody against S . agalactiae [20] and supports growth of S . agalactiaeto high cell densities [14, 66] . As the present study demonstratesan important role of DpsA for growth of S . agalactiae in amniotic fluid, DpsA represents an interesting target for the preventionor treatment of in utero S . agalactiae infections.

In several organisms, the expression of genes encoding peptide permeases is induced by the presence of peptides [7, 25, 44]or repressed in the absence of substrate [39, 57] . As shown by quantitative real-time PCR, the transcription of the S . agalactiae genes dppA to dppE was not affected in di- or hexapeptide-containingdCDM . However, the expression of the dppA-E operon increasedabout twofold during growth of the bacteria in THY medium, whichis a complex mixture of free amino acids, dipeptides, and oligopeptides.This indicates that components in THY medium, putatively peptides,cause an induction of dppA-E gene expression in S . agalactiae.In contrast, the expression of the dppA gene in E . coli is repressedduring growth of the bacteria in a peptide-rich complex medium[39] . In S . pyogenes as well, the transcription of the dppA-E operon is down-regulated during growth of the bacteria in THY medium [44] . This suggests different regulatory mechanisms in expression control of the dppA-E operon among these microorganisms.As demonstrated by our studies, the expression of the oppA1-Foperon was induced about eightfold in dCDM containing the hexapeptideVal-Tyr-Ile-His-Pro-Phe . However, no induction of oppA1-F expressionwas observed during growth of S . agalactiae in THY complex medium,which also contains dipeptides and free amino acids in additionto oligopeptides . It can therefore be speculated that in S. agalactiae the transcription of the oppA1-F operon is repressedby the presence of free amino acids and/or dipeptides in themedium . In L . lactis, the expression of the oppA gene is alsosignificantly repressed during growth of the bacteria in a complexmedium that is rich in free amino acids and peptides [13], indicatinga similar transcription control of the oppA-F operons in thesebacteria . In our studies, the expression of the dpsA gene wasnot altered during growth of the bacteria in different media,whereas in L . lactis, transcription of the dpsA ortholog dtpTis regulated by the peptide content of the medium [22] . Takentogether, our findings suggest that the transcriptional regulationof genes encoding peptide permeases in S . agalactiae is complexand partially distinct from the regulatory mechanisms of peptidepermease-encoding genes in other microorganisms.

In several gram-positive bacteria, peptide uptake systems arenot only essential for nutrient accumulation but they are alsoinvolved in determining other cellular functions, includingvirulence mechanisms . In pneumococci and S . pyogenes, the oligopeptide permease modulates the adherence of the bacteria to human hostcells [11, 12] . As revealed by our studies, the oligopeptidepermease from S . agalactiae also stimulates the adherence ofthe bacteria to human epithelial cells and modulates their bindingto fibrinogen and fibronectin . In addition, the oligopeptidepermease was shown to control the expression of the fbsA gene,which encodes a fibrinogen-binding adhesin . Similarly, the oligopeptidepermease from S . gordonii modulates the expression of the cshAgene, encoding a 259-kDa protein that mediates the binding ofthe bacteria to immobilized fibronectin and to other microorganisms[33, 34] . In S . pyogenes, the oligopeptide and the dipeptidepermease both control the transcription of the speB gene, encodinga cysteine protease which is important for the virulence ofthe bacteria [30, 44, 45] . Furthermore, in Bacillus cereus andBacillus thuringiensis, the oligopeptide permease controls theexpression of the plcR regulon, which is essential for the virulenceof these bacteria [19, 58] . As demonstrated by these examples, oligopeptide permeases are involved in the expression controlof virulence genes in various organisms . At present, the molecularbasis for the oligopeptide permease-dependent regulation offbsA expression in S . agalactiae remains unknown . However, a putative quorum-sensing system was recently identified in S. agalactiae O90R [61] . Interestingly, the inactivation of thisquorum-sensing system also changed the fibrinogen-binding propertiesof the resultant mutant . As quorum sensing in gram-positivebacteria is mediated by small peptides, it is tempting to speculatethat in S . agalactiae the oligopeptide permease is involvedin the quorum-sensing-dependent regulation of fbsA expression.In fact, in several gram-positive bacteria, oligopeptide permeasesare involved in quorum-sensing-dependent gene regulation andcontrol cellular properties like sporulation in Bacillus subtilis[43, 51], development of competence in B . subtilis and S . pneumoniae [5, 42], and conjugation in E . faecalis [29] . All these studiespoint to an involvement of peptide permeases in the transportof signaling molecules that in turn modulate gene expressionby interacting with transcriptional regulators . The precisemechanism of oligopeptide permease-dependent gene regulationin S . agalactiae and the full complement of molecules that participatein this signaling process remain to be identified . Studies aretherefore under way to unravel peptides with signaling functionin S . agalactiae and to identify further genes revealing anoligopeptide permease-dependent expression.

 
* Corresponding author . Mailing address: Dept . of Microbiology and Biotechnology, University of Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Germany . Phone: 49-731-5024853 . Fax: 49-731-5022719 . E-mail: dieter.reinscheid@biologie.uni-ulm.de .

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