csrrs regulates group b streptococcus virulence gene … · 2017. 8. 15. · cate that the csrrs...

JOURNAL OF BACTERIOLOGY, Sept. 2009, p. 5387–5397 Vol. 191, No. 170021-9193/09/$08.00�0 doi:10.1128/JB.00370-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

CsrRS Regulates Group B Streptococcus Virulence Gene Expression inResponse to Environmental pH: a New Perspective on

Vaccine Development�†Isabella Santi,1 Renata Grifantini,1 Sheng-Mei Jiang,2 Cecilia Brettoni,1 Guido Grandi,1*

Michael R. Wessels,2 and Marco Soriani1

Novartis Vaccines and Diagnostics Srl, Via Fiorentina 1, 53100, Siena, Italy,1 and Division of Infectious Diseases,Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts 021152

Received 17 March 2009/Accepted 15 June 2009

To identify factors involved in the response of group B streptococci (GBS) to environmental pH, weperformed a comparative global gene expression analysis of GBS at acidic and neutral pHs. We found that thetranscription of 317 genes was increased at pH 5.5 relative to that at pH 7.0, while 61 genes were downregu-lated. The global response to acid stress included the differential expression of genes involved in transport,metabolism, stress response, and virulence. Known vaccine candidates, such as BibA and pilus components,were also regulated by pH. We observed that many of the genes involved in the GBS response to pH are knownto be controlled by the CsrRS two-component system. Comparison of the regulon of wild-type strain 2603 V/Rwith that of a csrRS deletion mutant strain revealed that the pH-dependent regulation of 90% of the down-regulated genes and 59.3% of the up-regulated genes in strain 2603 V/R was CsrRS dependent and that manyvirulence factors were overexpressed at high pH. Beta-hemolysin regulation was abrogated by selective inac-tivation of csrS, suggesting the implication of the CsrS protein in pH sensing. These results imply that thetranslocation of GBS from the acidic milieu of the vagina to the neutral pH of the neonatal lung signals theup-regulation of GBS virulence factors and conversion from a colonizing to an invasive phenotype. In addition,the fact that increased exposure of BibA on the bacterial surface at pH 7.0 induced opsonophagocytic killingof GBS in immune serum highlights the importance of pH regulation in the protective efficacy of specificantibodies to surface-exposed GBS proteins.

Group B Streptococcus (GBS) is the leading cause ofneonatal septicemia and meningitis (52), and it has recentlybeen recognized as an increasingly common cause of inva-sive disease in nonpregnant adults (15). GBS is a commoninhabitant of the human gut and asymptomatically colonizesthe vaginas of one-third of women (23). Although GBS isconsidered mainly an extracellular pathogen, it has beenreported to be able to persist inside macrophages by impair-ing protein kinase C signal transduction (11). Taken to-gether, these observations suggest that, within the humanhost, GBS encounters pH conditions that vary from theacidic pH of the vagina or intracellular endocytic compart-ments to the near-neutral pH of amniotic fluid or the fetallung (5, 42). Accordingly, we hypothesized that GBS adaptsto different environmental pH conditions by modulating thetranscription of genes involved in pathogen-host interaction.Several studies indicate that the adherence of GBS to bothrespiratory and vaginal epithelial cells is enhanced at acidicpH, perhaps as a result of altered expression of surface-asso-ciated adhesins that favor colonization (6, 30, 62). In addition,it has been reported that gram-positive bacteria, such as oralstreptococci and lactic acid bacteria, can modify the expression

of their genes in response to pH shifts, activating several mech-anisms of acid resistance (12).

An important transcriptional regulatory system used bypathogenic streptococci to adapt to host conditions is the two-component regulatory system CsrRS (for capsule synthesis reg-ulator, regulator and sensor components; also called CovRS)(18, 31). In group A Streptococcus (Streptococcus pyogenes, orGAS), CsrRS is a major global regulatory system that canrepress as many as 15% of chromosomal genes, including anumber of surface-associated and secreted proteins that me-diate virulence and host-pathogen interactions (3, 16, 18, 33,39). Mg2� and the human cathelicidin antimicrobial peptideLL-37 have been identified as opposing signals that act throughCsrS to regulate an extensive repertoire of GAS genes (17,20–22). An orthologue of the CsrRS system has been identifiedin GBS (27, 31). Expression profiling studies suggest that asmany as 7% of GBS genes are regulated by CsrRS (28, 31).The CsrRS regulon includes genes that encode secreted orsurface components and transport systems for various smallmolecules, including peptides, amino acids, sugars, and metals.However, as opposed to GAS, where CsrR has been reportedto function almost exclusively as a repressor, in GBS somegenes are upregulated (28, 31). Studies of rodent models indi-cate that the CsrRS system plays a role in GBS virulence (31)(28). Moreover, Jiang et al. showed that distinct patterns ofgene regulation in CsrR versus CsrS mutants were associatedwith different hierarchies of relative virulence in murine mod-els of systemic infection and septic arthritis (28).

In this study, we report a genomewide transcriptional anal-

* Corresponding author. Mailing address: Novartis Vaccines, ViaFiorentina 1, 53100, Siena, Italy. Phone: (39) 0577 243390. Fax: (39)0577 243564. E-mail: [email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 19 June 2009.

5387

on August 15, 2017 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

ysis of GBS responses to acidic versus neutral pH conditionsand demonstrate that genes involved in virulence and adapta-tion to the host are modulated in response to environmentalpH. We found that CsrRS is necessary for the majority of thepH-dependent transcriptional changes, suggesting that CsrRShas a primary role in the adaptation of GBS to different pHenvironments.

Analysis of the GBS transcriptional response to changes inpH provides new insight into the mechanisms used by thispathogen to adapt to and colonize different anatomical micro-environments. In particular, the transition from colonization ofthe vagina to infection of the fetus or neonate is associatedwith a change in environmental pH that results in a strikingCsrRS-mediated up-regulation of virulence determinants im-plicated in invasive infection. Furthermore, the fact that tran-scriptional regulation of known vaccine candidates, includingthe GBS immunogenic bacterial adhesin BibA and pilus com-ponents, is pH dependent may open new perspectives in GBSvaccine development.

MATERIALS AND METHODS

Bacterial strains and growth conditions. GBS type V strain 2603 V/R, type Iastrain 515, mutant strains 515�csrS and 2603�csrS, and type III strain COH1have been described previously (27, 36, 56, 60). Escherichia coli DH10BT1 wasused for cloning purposes. Unless otherwise specified, for experiments testing theeffects of pH, GBS was cultured at 37°C in complex medium (10 g/liter proteosepeptone, 5 g/liter Trypticase peptone, 5 g/liter yeast extract, 2.5 g/liter KCl, 1 mMurea, 1 mM arginine). For experiments comparing wild-type GBS with �csrSmutants, GBS strains were grown in Todd-Hewitt broth (THB) (Difco) or onTrypticase soy agar supplemented with 5% defibrinated sheep blood (PMLMicrobiologicals). When appropriate, antibiotics were used at the followingconcentrations: for E. coli, erythromycin at 150 �g ml�1 and kanamycin at 50 �gml�1; for GBS, erythromycin at 1 �g ml�1 and kanamycin at 1,000 �g ml�1. E.coli was grown in Luria-Bertani broth.

Construction of a 2603 V/R csrRS deletion mutant. The csrRS gene was deletedin GBS 2603 V/R according to the procedure previously described (32). Thein-frame deletion fragment was obtained by splicing overlap extension PCRusing primers 5�-CGGGGTACCTGCCACGCGTTTTCATTGTT-3�, 5�-ATGAATTCAGTCCC TCTACACCAACCTTCTTATTC-3�, 5�-AGAAGGTTGGTGTAGAGGGACTGAATTCATTGTTC-3�, and 5�-CGCGCGGATCCTTCAAGGAACACTAGAGCAC-3�. KpnI and BamHI restriction enzyme cleavage siteswere incorporated at the 5� ends of primers (underlined and bold sequences,respectively) in order to clone the fragment into the KpnI-BamHI-digestedpJRS233 plasmid, which was a gift from June Scott (45). After the in-framedeletion fragment was cloned into pJRS233, plasmid pJRS233�csrRS was ob-tained. Plasmid pJRS233�csrRS was then transformed into strain 2603 V/R byelectroporation, and transformants were selected after growth at 30°C on agarplates containing 1 �g ml�1 erythromycin. Transformants were then grown at37°C with erythromycin selection as previously described (4). Integrant strainswere serially passaged for 5 days in liquid medium at 30°C without erythromycinselection to facilitate the excision of the plasmid, resulting in the csrRS deletionon the chromosome. Dilutions of the serially passaged cultures were plated ontoagar plates, and single colonies were tested for erythromycin sensitivity to con-firm the absence of pJRS233 sequences.

Construction of Pcyl-lacZ transcriptional fusions and �-galactosidase assay.The promoter of the cyl operon was PCR amplified with two specific primers thatcarried an EcoRI or BamHI restriction enzyme site at the 5� end. The PCRproduct was digested with EcoRI and BamHI and was cloned into vector pTCV-lac immediately upstream of the promoterless lacZ gene (48). The plasmidconstruct harboring lacZ fusions (pTCV-Pcyl-lacZ) was introduced into GBSstrains 515 and 515�csrS mutant by electroporation, and transformants wereselected by growth at 37°C in the presence of erythromycin. GBS strains weregrown in THB to mid-exponential phase (cell density, 3 � 108 CFU/ml). Cellswere collected and then permeabilized by treatment with 0.5% toluene and 4.5%ethanol (48). �-Galactosidase activity was determined as described by Miller(40). Briefly, a 0.1-ml aliquot of the treated culture was added directly to 0.9 mlZ buffer (60 mM Na2HPO4 · 7H2O, 40 mM NaH2PO4 · H2O, 10 mM KCl, 1 mMMgSO4 · 7H2O, 50 mM �-mercaptoethanol). The suspension was preincubated

for 5 min at 28°C and then incubated with 0.2 ml of 4-mg/ml 2-nitrophenyl-�-D-galactopyranoside. Reactions were stopped by the addition of 0.5 ml of 1 MNa2CO3. Activity was expressed as [103 � (OD420 of the reaction mixture � 1.75OD550 of the reaction mixture)] divided by [time of the reaction (in minutes) �OD600 of the quantity of cells used in the assay], where OD420 is the opticaldensity at 420 nm.

Hemolysin assay. Approximately 108 CFU of GBS cells was collected fromliquid cultures at exponential phase, washed once with phosphate-buffered saline(PBS), and resuspended in 1 ml of PBS with 0.2% glucose. Serial dilutions of thissuspension in 1� PBS–0.2% glucose were mixed with an equal volume of 1%sheep erythrocytes in the same buffer and incubated at 37°C for 1 h. Afterincubation, unlysed erythrocytes and bacteria were removed by centrifugation,and the hemoglobin content of the supernatant was assessed by measuring theA405. The hemolytic titer of each strain was determined as the reciprocal of thegreatest dilution producing 50% hemoglobin release compared with controlsamples in which all erythrocytes were lysed by 1% sodium dodecyl sulfate (27).

Microarray procedures and data analysis. The GBS DNA microarray wasprepared by amplifying 250- to 500-bp PCR fragments of each annotated openreading frame from genomic DNA of GBS strain 2603 V/R (56). Microarraycomparison was performed on the wild-type strain 2603 V/R and the isogenicmutant strain 2603�csrRS grown at pH 7.0 to late-exponential phase and thenresuspended at an OD650 of 0.3 to 0.4 at pH 7.0 or pH 5.5 for 30 min at 37°C. Nobacterial growth was observed during the 30 min of incubation. For each strain,total bacterial RNA was extracted from four independent culture pools and wasused for cDNA synthesis and labeling. In each experiment, the two RNA samplesto be compared were converted to cDNA, fluorescently labeled with Cy3 or Cy5in direct (Cy3–Cy5) and dye swap (Cy5–Cy3) labeling reactions to control fordye-dependent variations in labeling efficiency, and cohybridized on the arrays.Labeling (dye and dye swap) and hybridization reactions were carried out induplicate, and therefore a total of four arrays were used for each pairwisecomparison. Probe hybridization and washing were performed as recommendedby the slide supplier. Slides were scanned with a ScanArray 5000 instrument(Perkin-Elmer) at a resolution of 10 �m per pixel. The resulting 16-bit imageswere processed using the Imagene program (BioDiscovery). For each image, thesignal value of each spot was determined by subtracting the mean pixel intensityof the background and normalizing according to the Lowess normalizationmethod. The fluorescence intensity of each gene obtained from the four inde-pendent array hybridizations represents the mean of 16 individual hybridizations,because each gene probe is represented four times on each array. For each gene,the difference in expression was calculated as the ratio of the fluorescence signalsof the two cDNA samples compared. The statistical significance of the differencein mean fluorescence intensity calculated for each gene between the two RNAsamples compared was determined by an unpaired two-tailed Student t test. Thenull hypothesis was rejected, because the false discovery rate, estimated by theprocedure described by the National Institute of Aging array analysis tool,was less than 0.02. Genes whose expression ratios exceeded twofold at a Pvalue of �0.01 were considered differentially expressed.

Quantitative reverse transcription-PCR (qRT-PCR). GBS strains grown over-night on Trypticase soy blood agar plates were inoculated in 10 ml THB (pH 5.0or pH 7.4). The GBS cells were collected at the mid-exponential-growth phase(OD650, 0.3) by centrifugation (3,200 � g, 5 min). The pellet was resuspended in0.5 ml 0.9% NaCl and 1 ml RNA Protect buffer (Qiagen) and was kept at roomtemperature for 5 min. After centrifugation, the bacterial pellet was treated with100 U mutanolysin (Sigma) and 15 mg/ml lysozyme (Sigma) in Tris-EDTAbuffer, pH 8.0, in a final volume of 100 �l. Total bacterial RNA was then isolatedusing an RNeasy minikit (Qiagen) according to the manufacturer’s instructions.RNA samples were treated with DNase I (Invitrogen) for 30 min at 37°C toremove DNA contamination. The RNA concentration was adjusted to 100 ng/�l,and samples were stored at �80°C until use (28).

Purified RNA and gene-specific primer pairs were used for real-time PCRassays using SYBR green reagents (Qiagen) and an ABI 7000 sequence detectioninstrument (Applied Biosystems) according to the manufacturer’s instructions.Duplicate reaction mixtures containing 50 ng of template RNA were performedfor the genes of interest. To exclude DNA contamination of RNA samples,replicate control samples in which reverse transcriptase was omitted were as-sayed. Standard curves were obtained by performing PCR with SYBR greendetection on serial dilutions of spectrophotometrically quantified genomic DNA.The housekeeping gene recA was used as a reference control gene to normalizeexperimental results. The following conditions were used for the real-time PCR:1 cycle for 30 min at 50°C, 1 cycle for activation for 15 min at 95°C, and 36 cyclesof PCR with denaturation at 95°C for 15 s, annealing for 30 s at 55°C, andextension for 30 s at 72°C. Relative gene expression was quantified by the

5388 SANTI ET AL. J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

comparative threshold cycle method (��CT method) according to the manufac-turer’s guidelines (Applied Biosystems).

Bacterial extracts and immunoblot analysis. GBS protein extracts were pre-pared by growing bacteria at pH 7.0 or pH 5.5 at 37°C. Aliquots (10 ml) ofbacteria were collected at different time points, washed in PBS, and incubated for2 h at 37°C in 200 �l of 50 mM Tris-HCl (pH 6.8) containing protease inhibitors(Roche Diagnostic) and 400 U/ml of mutanolysin (Sigma, St. Louis, MO). Bac-terial cells and particulates were removed by centrifugation, and the superna-tants containing peptidoglycan-associated proteins were used for immunoblotanalysis. Immunoblot analysis of pH-regulated proteins and BibA was performedby transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis to nitrocellulose membranes (Protran). The membranes werethen blocked with 5% milk in 0.1% PBS–Tween 20 and overlaid for 1 h withspecific mouse or rabbit antisera diluted 1:1,000 in the same buffer. After a wash,membranes were incubated with the respective horseradish peroxidase-conju-gated secondary antibodies diluted 1:1,000, and detection was performed byenhanced chemiluminescence.

Flow cytometry analysis. In order to quantify the exposure of BibA on thebacterial surface, GBS was grown at pH 7.0 or pH 5.5 at 37°C. Aliquots ofbacteria at different time points were fixed with 2% paraformaldehyde for 20 minat room temperature and were then incubated with rabbit anti-BibA serum.Bacteria were then washed and incubated with R-phycoerythrin-conjugated sec-ondary antibodies (Jackson ImmunoResearch, West Grove, PA) at 4°C. After awash, bacteria were analyzed with a FACSscan flow cytometer (Becton Dickin-son). Data were analyzed with the FlowJo program (version 7.2.2).

Opsonophagocytosis assay. The functional capacity of antibodies to bind toBibA protein on the GBS surface was assessed by measurement of in vitro killingin an opsonophagocytosis assay, as reported previously (34). In brief, the reac-tions took place in a total volume of 125 �l containing 2 � 106 human polymor-phonuclear leukocytes (PMNs), 2 �105 CFU of GBS cells, 10% baby rabbitcomplement (Cedarlane), and heat-inactivated mouse antisera diluted 1/30 at37°C for 1 h with shaking at 350 rpm. Immediately before and after 1 h ofincubation, a 25-�l aliquot was diluted in sterile distilled water and plated ontoTrypticase soy agar plates with 5% sheep blood. A set of negative controlsincluded in each experiment consisted of reaction mixtures containing a serumderived from mice immunized with PBS, reaction mixtures without PMNs, andreaction mixtures with heat-inactivated complement.

Microarray data accession number. The microarray experiment has beensubmitted to the Array Express database of the European Bioinformatic Institute(http://www.ebi.ac.uk/microarray-as/ae/) with accession number E-TABM-708.

RESULTS

Regulation of GBS gene expression by pH. To characterizethe genomewide regulation of GBS gene transcription in re-sponse to environmental pH, we compared the transcriptomechanges induced in strain 2603 V/R by exposure to pH 5.5 withglobal gene expression at pH 7.0. Briefly, the experimentalprotocol consisted in growing bacteria at pH 7.0 (37°C) tolate-exponential phase, followed by incubation at pH 7.0 or pH5.5 for an additional 30 min. These conditions did not affectcell viability; after exposure to pH 5.5, the number of CFU wasunchanged from that at pH 7.0 (data not shown). BacterialRNA was purified, converted to fluorescently labeled cDNA byreverse transcription, and hybridized to a GBS genomic DNAmicroarray. Tables S1 and S2 in the supplemental material reportthe differentially expressed genes, i.e., those that showed a statis-tically significant change (P � 0.01) of at least twofold in thetranscript amount. Exposure of GBS to an acidic pH had signif-icant and widespread effects on global gene expression. Indeed,we observed that the expression of 378 genes (18% of the ge-nome) was altered from that at pH 7.0 during exposure to pH 5.5for 30 min. Upregulation at pH 5.5 was more prevalent, involving317 genes (83.9%), while 61 genes (16.1%) were downregulated.Many of the regulated genes are adjacent to one another on thechromosome and are likely to represent polycistronic operonsencoding proteins with related functions.

Genes related to protein synthesis were among the mostregulated gene families (Fig. 1), consistent with a requirementfor de novo protein synthesis for adaptation to an acidic envi-ronment (24). In addition, genes encoding transport and bind-ing proteins were regulated at an acidic pH, with a total of 50genes upregulated and only 2 downregulated (SAG1467 andSAG1813). A list of the most highly regulated genes at an

FIG. 1. Differential regulation of gene expression in GBS strain 2603 V/R after exposure to pH 5.5. or pH 7.0. Genes were classified into 17functional categories. Bars indicate the numbers of genes differentially regulated at pH 5.5 versus pH 7.0.

VOL. 191, 2009 RESPONSE OF GBS TO pH VARIATION 5389


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

acidic pH is reported in Table 1. The microarray data werevalidated by qRT-PCR on four genes, using total RNA isolatedfrom two independent cultures of wild-type and csrRS mutantbacteria grown under the two pH conditions (see Table S3 inthe supplemental material). The changes in response to the pHshift observed in wild-type GBS and in the mutant strain werevery similar to those measured by the microarray, even thoughthe primer pairs used for qRT-PCR amplified gene regionsdifferent from those spotted on the microarray. The microarraydata were further confirmed by immunoblot analysis on cellextracts of GBS grown at pH 7.0 or pH 5.5 (see Fig. S1 in thesupplemental material).

Transport genes. Several genes encoding transport proteinswere upregulated at pH 5.5. These included fhuD (SAG1393)and fhuB (SAG1394) of the fhuCDBG operon, which codes fora siderophore-dependent iron transporter (10); the SAG1007-to-SAG1009 operon, whose homologous genes in Streptococ-cus mutans are involved in iron uptake and transport (1); mtsC(SAG1532) and mtsB (SAG1531), homologous to the GASmtsABC operon; and SAG0745, coding for a putative NRAMPfamily transporter involved in both ferric and manganeseion uptake (25, 43). We also observed the fourfold up-regulation of SAG1711, coding for a putative CorA proteininvolved in magnesium transport (58). Other pH-up-regu-lated genes of this family are involved in amino acid orpeptide transport, including the dppABCDE operon(SAG0187 to SAG0191), which encodes a dipeptide ABCtransporter (SAG0188), and the dpsA gene (SAG1444), cod-ing for a putative proton-driven symporter (49). Among theup-regulated genes, we also found a putative methionineABC transporter (SAG1638, SAG1639, and SAG1641) sim-ilar to S. mutans AtmBDE (53); a putative histidine ABCtransporter (SAG0947 to SAG0949); a spermidine-putrescinetransporter, PotABCD (SAG1108 to SAG1111); and thehighly regulated SAG0290-to-SAG0292 operon (11-fold to 23-

fold increased), encoding a putative polar amino acid ABC trans-porter. In addition, SAG0241-SAG0242-SAG0244, encodingcomponents of the glycine/betaine osmoregulation system,which is reported to mediate the adaptation of Bacillus subtilisand Lactococcus lactis to osmotic stress, was up-regulated atpH 5.5 (29, 41). Finally, the pstBC genes, involved in phosphatetransport (SAG0988 and SAG1965), and PhoU (SAG0987),which regulates phosphate uptake, were also up-regulated.

Metabolism genes. Folate biosynthesis pathway genes (operonSAG1114-SAG1116-SAG1117) were up-regulated at an acidicpH, while glnA (SAG1763), a gene involved in the synthesis ofglutamate, was down-regulated under the same conditions. Inagreement with the latter result, glnR (SAG1764), encoding atranscriptional regulator implicated in the modulation of glu-tamine and glutamate metabolism, was fivefold down-regu-lated. Moreover, SAG2125 and SAG2126, genes encoding acarbamate kinase and an ornithine carbamoyltransferase, re-spectively, were highly up-regulated (Table 1). Both enzymesare components of the arginine deiminase system, which hasbeen suggested to aid bacterial survival in acidic environmentsby catalyzing the release of ammonia from arginine (19).

Stress response genes. Among stress response genes, tran-script changes were observed at pH 5.5 in the class I heat shockoperon hrcA-grpE (SAG0095-SAG0098), which was down-reg-ulated 3.7- to 7-fold, in contrast to what has been observed forother bacteria during acid stress (26). sodA (SAG0788), a genethat confers protection against oxidative stress and enhancesbacteremia in mice (47), was also less expressed at pH 5.5.Other genes encoding cold shock-induced proteins, suchas deaD (SAG0777), coding for RNA helicase, and pnp(SAG0203), coding for polyribonucleotide nucleotidyltrans-ferase, were 4.3- and 2.3-fold up-regulated, respectively, at pH5.5. In addition, SAG0185 and SAG0186, genes encoding twoproteins similar to Staphylococcus aureus LrgAB, involved inthe regulation of murein hydrolase activity and cell death (2,

TABLE 1. List of genes highly regulated in GBS strain 2603 V/R after exposure to pH 5.5 versus pH 7.0

TIGR locus Annotation Common name Family Fold changea Regulation

SAG0008 Transcription-repair coupling factor mfd DNA metabolism 13.39 UpSAG0185 Hypothetical protein Cell envelope 22.85 UpSAG0186 Hypothetical protein Unknown function 17.97 UpSAG0290 ABC transporter, substrate-binding protein Transport and binding proteins 22.98 UpSAG0291 Amino acid ABC transporter, permease protein Transport and binding proteins 15.59 UpSAG0292 Amino acid ABC transporter, ATP-binding protein Transport and binding proteins 11.37 UpSAG0336 Helicase, putative Unknown function 10.10 UpSAG0662 CylX protein Cellular processes 26.00 DownSAG0663 CylD protein cylD Cellular processes 15.78 DownSAG0664 CylG protein cylG Cellular processes 17.93 DownSAG0665 Acyl carrier protein AcpC Fatty acid and phospholipid

metabolism10.49 Down

SAG0666 CylZ protein Cellular processes 28.83 DownSAG0667 CylA protein cylA Cellular processes 11.78 DownSAG0669 CylE protein cylE Cellular processes 11.03 DownSAG0670 CylF protein cylF Cellular processes 13.95 DownSAG0671 CylI protein cylI Cellular processes 11.88 DownSAG0672 CylJ protein cylJ Cellular processes 12.75 DownSAG0673 CylK protein cylK Cellular processes 15.20 DownSAG0723 RNase III rncS Transcription 11.48 UpSAG2063 Pathogenicity protein bibA Cellular processes 9.1 DownSAG2125 Carbamate kinase arcC-1 Energy metabolism 19.01 UpSAG2126 Ornithine carbamoyltransferase argF-1 Amino acid biosynthesis 13.73 Up

a P � 0.0001. Except for BibA, the selected genes had an expression ratio exceeding 10-fold.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

8, 44, 59), were 23- and 18-fold up-regulated, respectively(Table 1).

Transcriptional regulators. SAG2127, encoding a putativesensor histidine kinase, was upregulated 2.2-fold at pH 5.5.Other regulatory genes up-regulated at an acidic pH includedSAG0277, encoding an Mga-like protein implicated in sugarmetabolism; adcR (SAG0154), encoding the regulator of theAdcCBA high-affinity zinc uptake system; rbsR (SAG0119),coding for the ribose operon repressor; SAG0251, SAG2015,and SAG2037, putative Cro/CI family regulators; SAG0427, aputative MerR family regulator; SAG0796, a putative MarRfamily regulator; and SAG0327, coding for an uncharacterizedtranscriptional regulator.

Virulence and host-pathogen interaction genes. Transcrip-tion of several known or putative virulence factors was differ-entially regulated in response to pH. Expression of the cyl genecluster (SAG0662 to SAG0673), required for GBS hemolysinproduction, was markedly higher at a neutral pH (9- to 33-fold)than at acidic pH. A similar pattern of expression was observedfor several surface-expressed proteins containing the LPXTG cellwall-sorting motif: bibA (SAG2063), which encodes an antiphago-cytic surface protein (50); SAG0416, encoding a predicted proteinwith 67% amino acid identity to C5a peptidase; and SAG0677,

which encodes an LPXTG-containing protein of unknown func-tion. In contrast, cfb (SAG2043), encoding CAMP factor, and asecond gene, SAG2042, transcriptionally linked to cfb and pre-dicted to encode a rhodanese-like protein of unknown func-tion, were both down-regulated at pH 7.0. Similarly, genesbelonging to pilus island I (SAG0646 to -0648) were morehighly expressed at pH 5.5 than at pH 7.0 (see Tables S1 andS2 in the supplemental material). Genes involved in bacterialmetabolism have been associated with GBS pathogenesis, andin this context we observed up-regulation at pH 5.5 of theSAG1739-to-SAG1744 operon, encoding proteins involved inrespiration metabolism and important for GBS growth in vivoand virulence (61). Other genes involved in both metabolismand virulence that were up-regulated at pH 5.5 include thedppABCDE operon, containing a dipeptide ABC transporter(2.48-fold), and dpsA, reported to be important in the bindingof GBS to host proteins, in attachment to eukaryotic cells, andin the expression of virulence genes (49); potABCD, implicatedin the pathogenesis of Streptococcus pneumoniae in variousinfection models (57); and glnQ, the glutamine transport generequired both for adherence to fibronectin in vitro and forvirulence in vivo (55).

Interestingly, a number of known vaccine candidates were

FIG. 2. Differential regulation of gene expression in GBS strain 2603 V/R versus the isogenic �csrRS mutant strain after exposure to pH 5.5compared to pH 7.0. Open bars indicate the number of pH-regulated genes in the wild-type strain; shaded bars indicate the number of genes thatare pH dependent and CsrRS dependent; filled bars indicate the number of genes that are pH dependent and CsrRS independent.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

found among the pH-dependent genes (see Table S1 in thesupplemental material), including genes encoding pilus com-ponents (34), BibA (49a), Sip (7), and C5a-peptidase (54).

The majority of pH-dependent transcriptional changes arecontrolled by CsrRS. The microarray analysis revealed that 43genes previously reported to be modulated by CsrRS (28, 31)were among the pH-regulated genes. These included geneshighly regulated by the CsrRS system in various GBS strains,such as SAG0416 (encoding a protein similar to C5a pepti-dase), SAG0662 to SAG0673 (cyl operon), SAG2042 (encod-ing a rhodanese-like protein), SAG2043 (cfb), and SAG2063(bibA). To test whether pH-dependent regulation of gene expres-sion could be mediated through the CsrRS system, we con-structed a csrRS in-frame deletion mutant strain in the 2603 V/Rbackground and analyzed its transcriptome profile under the

same conditions used for the wild-type strain. Exposure of the�csrRS mutant strain to pH 5.5 compared to pH 7.0 was associ-ated with changes in gene expression that were less widespreadand generally smaller in magnitude than those in the wild type,with only 126 genes regulated compared to the 378 genes mod-ulated in the wild-type strain. The pH-dependent regulationfound by microarray analysis was verified by qRT-PCR for fourrepresentative genes in both the wild-type strain, 2603 V/R, andthe �csrRS mutant strain (see Table S3 in the supplemental ma-terial). Of the genes that were differentially expressed at pH 5.5 inthe wild-type strain, 91% of the down-regulated genes and 63% ofthe up-regulated genes were not differentially regulated at anacidic pH in the �csrRS mutant, indicating that these genes areboth pH and CsrRS dependent (see Table S1 in the supplementalmaterial). The fact that the majority of down-regulated geneswere CsrRS dependent is in agreement with earlier observationsthat CsrR acts predominantly as a repressor (28, 31). Among thegenes down-regulated at pH 5.5, we found a number of factorsinvolved in GBS metabolism, transport, and virulence (Fig. 2).These data strongly suggest that CsrRS is involved in the responseof GBS to an acidic pH. In addition to genes that appeared to bepH and CsrRS dependent, we found a group of 94 genes (24.9%)modulated by pH both in the wild-type strain and in the �csrRSstrain, indicating that their expression was pH dependent andCsrRS independent (see Table S2 in the supplemental material).Only six genes were down-regulated in a CsrRS-independentmanner, including the hrcA-grpE operon, while among the up-regulated genes, we found genes encoding proteins involved inprotein synthesis and in purine and pyrimidine synthesis, thetranscriptional regulator AraC, proteins involved in nutrienttransport, and the LrgAB system.

FIG. 3. pH-dependent regulation of beta-hemolysin expression in GBS strain 515 requires CsrS. (A and B) Beta-galactosidase activity(reflecting transcription from the cyl operon promoter) in GBS reporter strain 515/pTCV-Pcyl-lacZ (A) or in 515�csrS/pTCV-Pcyl-lacZ (B) grownat pH 5.0 to 7.5. Data are representative of two independent experiments. (C and D) Hemolytic activity of GBS wild-type strain 515 (C) or 515�csrS(D) grown at pH 5.0 to 7.5. Values are means standard deviations from at least three independent experiments.

TABLE 2. Effects of pH on the expression of cylE and scpB inGBS wild-type and csrS mutant strains

GBS strain

Fold change in gene transcription (pH 7.4/pH 5.0)a

cylE scpB

2603 V/RWild type 17.6 4.8 7.6 3.9�csrS 0.6 0.2 0.9 0.7

515 IaWild type 21.5 3.5 8.0 4.2�csrS 0.24 0.0 0.7 0.4

a Data are expressed as the ratio of the amount of transcript detected in the GBSstrain grown at pH 7.4 to that detected in the same strain grown at pH 5.0. Valuesare means standard deviations from at least three independent experiments.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Finally, 32 genes were found to be differentially expressedin response to pH only in the �csrRS mutant. Some of them(25%), although they did not reach our threshold to be con-sidered differentially expressed, showed a trend toward regu-lation in the wild type (change, 1.6-fold; P � 0.01), indicatingthat they might belong to the pH-dependent and CsrRS-inde-pendent group. The remaining genes are likely to have more-complex regulation mechanisms whose elucidation will requirefurther investigation.

pH regulation of CsrRS-dependent genes requires the CsrSsensor protein. The data described above establish a strongassociation between pH regulation of GBS gene expressionand the CsrRS two-component system: the majority of pH-regulated genes in the wild-type strain were not similarly reg-ulated in the �csrRS mutant. These results suggest that anacidic environmental pH might signal through the CsrS sensorprotein to induce the autokinase activity of its cytoplasmicdomain and subsequent phosphotransfer to the CsrR regula-tor, thereby increasing its activity as a transcriptional regulatorof target promoters. At a neutral pH, the autokinase activity ofCsrS would not be stimulated, and CsrR would remain unphos-phorylated and relatively inactive as a transcriptional regula-tor. To investigate more directly the role of the CsrS sensorprotein in pH regulation, we compared the wild-type strain,2603 V/R, and an isogenic csrS mutant with respect to expres-sion of two CsrRS-regulated virulence genes implicated ininvasive infection: cylE, encoding beta-hemolysin, and scpB,encoding C5a peptidase. In order to model the pH environ-ments of peripartum infection, we tested gene expression atpH 5.0 (to mimic the maternal birth canal) and pH 7.4 (torepresent the lungs or bloodstream of the fetus or newborn).qRT-PCR of transcripts for cylE and scpB revealed a strikingincrease in expression at pH 7.4 over that at pH 5.0 in wild-typestrain 2603 V/R, but not in 2603�csrS (Table 2). We obtainedsimilar results when we compared cylE and scpB gene expres-sion in an isogenic csrS mutant versus the wild type in thebackground of GBS strain 515, a serotype Ia strain typical ofclinical isolates associated with neonatal sepsis, indicating thatthe CsrS regulation mechanism is conserved in different GBSstrains (60). We constructed reporter strains in the backgroundof GBS strain 515 in order to analyze in more detail thepH-dependent regulation of the cyl operon. For this purpose,the cyl operon promoter was fused to a promoterless genesequence encoding beta-galactosidase in an E. coli-streptococ-cal shuttle vector. The reporter construct was introduced intowild-type GBS strain 515 and into 515�csrS. Cell lysates wereprepared from each reporter strain after growth in a mediumat a range of pH values from pH 5.0 to pH 7.5. A gradedincrease in beta-galactosidase activity was observed with in-creasing pH for wild-type strain 515 but not for 515�csrS (Fig.3A and B). These results further confirmed that pH regulationof hemolysin expression requires CsrS and is mediated throughtranscription from the cyl operon promoter. We also tested thehemolytic activity of GBS grown at a range of pH values. Theseexperiments demonstrated the expected pH-related increase incytolytic activity for wild type strain 515 but not for 515�csrS(Fig. 3C and D).

BibA expression is reversibly regulated by the environmen-tal pH. To better define the relationship of pH to CsrRS-mediated gene regulation, we analyzed in greater detail the

effect of pH on the expression of bibA, one of the most highlyregulated genes, with a 10-fold increase in expression at pH 7.0over that at pH 5.5. During growth at pH 7.0, bibA geneexpression was maximal by RT-PCR at mid-exponential phase(2 h after the initial inoculum), after which it declined to aplateau level (Fig. 4A). As expected, at pH 5.5 the levels ofbibA expression were very low throughout the time span of theexperiment (Fig. 4A). No significant differences were observedin the growth rate, viability, or cell density reached at station-ary phase (data not shown). Immunoblot analysis confirmedthat the BibA protein is highly expressed at pH 7.0, while areduced amount of protein was observed at pH 5.5 (Fig. 4B).The SAG0392 protein, whose expression was not regulated bypH, was used as a loading control (Fig. 4B). In agreement withthese findings, flow cytometry analysis revealed that BibA ishighly exposed on the bacterial surface at pH 7.0, while at pH5.5 the protein is almost undetectable (Fig. 4C).

We also tested whether the level of expression of BibA on

FIG. 4. Effect of pH on bibA expression. GBS strain 2603 V/R wasgrown at pH 7.0 or pH 5.5 and was sampled every hour up to 4 h.(A) bibA gene expression was analyzed by RT-PCR on total RNAextracted under both pH conditions at different time points. (B) Im-munoblot analysis of BibA protein expression on bacterial extractsfrom GBS grown under different pH conditions. Immunoblotting ofprotein SAG0392, which is not regulated by pH, was used as a controlfor protein loading. (C) Graph reports flow cytometry analysis data,expressed as mean fluorescence intensity (MFI), for the time course ofBibA protein exposure on the surfaces of bacteria grown at pH 7.0 orat pH 5.5.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

the bacterial surface changes when the bacteria encounter anew pH environment. The wild-type strain was grown at pH 7.0or 5.5 and was then diluted in fresh medium at the other pH.The shift from a neutral to an acidic pH was associated with agradual decline in BibA surface expression (Fig. 5A), whereasthe transition from pH 5.5 to pH 7.0 resulted in a rapid in-crease, as confirmed by RT-PCR (Fig. 5B) and flow cytometryanalysis (Fig. 5C). These results further confirm pH regulationof BibA expression and show that up- or down-regulation isfully reversible upon exposure to a new pH environment. Sim-ilar results were obtained for differential BibA expression atpH 7.0 versus pH 5.5 for a panel of 11 clinical isolates of GBS(see Table S4 in the supplemental material), suggesting thatpH-dependent regulation is widespread among GBS strains.

Increased exposure of BibA on the bacterial surface at pH7.0 correlates with increased opsonophagocytic killing of GBS.One implication of the pH-dependent regulation of GBS sur-face proteins is that target antigens for specific antibodies maybe expressed only in a particular pH environment. Since thelevel of expression of an immunogenic antigen correlates withthe capacity of antisera to opsonize GBS (34), we testedwhether the upregulation of BibA surface expression at pH 7.0relative to pH 5.5 (Fig. 6A) correlated with increased antibody-mediated opsonophagocytic activity. We found that anti-BibAantibodies were able to induce complement-dependent op-sonophagocytic killing of GBS strain COH1 grown at pH 7.0,while no killing activity was observed against bacteria grown atpH 5.5 (Fig. 6B). An antiserum to the type III capsular poly-saccharide was used as a positive control. These findings high-light the importance of pH regulation in the protective efficacyof specific antibodies to GBS surface-exposed proteins.

DISCUSSION

The data reported in this paper provide evidence that GBSresponds to pH variations by activating a broad range of tran-scriptional changes involving genes linked to metabolism andvirulence. For the vast majority of these genes, regulation ofexpression in response to pH involves the CsrRS two-compo-nent system. In particular, we have observed that CsrRS isdirectly involved in the GBS response to acid stress. Recentreports have indicated that adaptation of GBS to both humanblood and a temperature shift induced the modulation of awide range of genes, mainly those involved in metabolism andvirulence (37, 38). The effect of pH on the GBS transcriptomepredicts a general trend of adaptive regulation during thegrowth of the organism in distinct anatomic sites that caninclude the acidic milieu of the vagina or the near-neutral pH

FIG. 5. Kinetics of reduction of BibA protein exposure on thebacterial surface at pH 5.5. (A) Bacteria were grown at pH 7.0 (timezero) and then shifted to pH 5.5 (filled circles) or kept at pH 7.0 (opencircles) for as long as 4 h. Data are represented as the percentages of

the mean fluorescence intensity (MFI) for BibA exposure at the dif-ferent time points relative to the MFI at time zero. Data are means standard deviations from at least three independent experiments. (B)Bacteria were grown at pH 5.5 and then incubated at pH 7.0 or at pH5.5 for 30 min. bibA gene expression was analyzed by RT-PCR on totalRNA extracted from both pH conditions. (C) Bars represent the timecourse of BibA protein exposure on the surfaces of bacteria grown atpH 5.5 and then shifted to pH 7.0 or kept at pH 5.5 for as long as 3 h.The red lines represent the growth curves of bacteria under the twodifferent experimental conditions, expressed as CFU ml�1.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

of amniotic fluid or the fetal lung during peripartum infection(51). In order to colonize the host successfully, GBS mustadapt quickly to changes in environmental pH by stimulatingcellular processes such as transcription and translation ofgenes involved in transport and metabolism. In line with thisconcept, we found that polyamine transport, known to be im-portant for the initial step of bacterial infection (57), is acti-

vated under acid stress. Transporters of amino acids, peptides,and metal ions were also upregulated under acidic conditions.Peptide uptake may be important not only for nutritional re-quirements under acid stress but also for bacterial attachmentto the vaginal epithelium. For example, in S. pneumoniae andGAS, the oligopeptide permease modulates the adherence ofthe bacteria to human host cells (13, 14), while in GBS it

FIG. 6. Enhanced exposure of BibA on the bacterial surface at pH 7.0 correlates with increased opsonophagocytic killing of GBS. (A) Flowcytometry analysis of GBS strain COH1 grown at pH 7.0 or at pH 5.5. Bacteria were incubated with a mouse antiserum against the GBS capsularpolysaccharide (�-Cps) (positive control), an antiserum from a mouse immunized with PBS alone (negative control), or a mouse antiserum againstBibA. Bacteria were then stained with an R-phycoerythrin-conjugated anti-mouse immunoglobulin G antibody (open histograms). Shadedhistograms represent bacteria incubated with the secondary antibody alone. (B) Live GBS bacteria were incubated for 1 h with human PMNs inthe presence of baby rabbit complement and specific antisera. The log10 of the difference between the bacterial CFU at time zero (t0) and at 1 h(t1) is shown. Positive values indicate bacterial killing, while negative values indicate net growth. Open bars represent GBS grown at pH 7.0; filledbars, GBS grown at pH 5.5. Anti-Cps and anti-PBS sera were used as positive and negative controls, respectively. Bacteria incubated without PMNsserve as a further control. Error bars indicate standard deviations.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

stimulates adherence to human epithelial cells and modulatestheir binding to fibrinogen and fibronectin (49).

The fact that responses of GBS to variations in pH in largepart required the CsrRS system led us to hypothesize thatduring GBS pathogenesis, CsrRS mediates the adaptation ofthe organism to the acidic environment of the vagina and tomore-basic compartments, such as the amniotic fluid and neo-natal lung. We postulate that the exposure of GBS to the acidicmilieu of the vagina induces the down-regulation of a numberof virulence factors and the upregulation of genes involved incolonization, including transporters and adherence factors im-portant for bacterial attachment to the epithelium. The adap-tive value of the pH response of GBS is further enhanced by itsreversibility, as demonstrated for bibA. The up-regulation ofvirulence factors such as BibA, beta-hemolysin, and C5a pep-tidase during passage from the acidic to the more neutral orbasic environment may be a critical factor in the invasive be-havior of GBS during peripartum infection of the neonate.

The mechanism by which pH variations modulate the ex-pression of CsrRS-regulated genes remains undefined. We ob-served no change in the transcript level of CsrRS at an acidicpH, suggesting that pH-dependent regulation is not a functionof changes in the abundance of the CsrRS proteins. However,it has been reported recently that, at least for S. mutans, theincrease in pH in the growth medium led to an increase in csrRexpression (9). An intriguing possibility suggested by our re-sults is that the increase in proton concentration at an acidicpH signals through the CsrS sensor protein to stimulate CsrSautokinase activity, with subsequent downstream phosphoryla-tion of CsrR, thereby increasing the affinity of CsrR for regu-lated promoters (28). Our finding that pH-dependent regula-tion of hemolysin and C5a peptidase is abrogated in csrSmutant strains is consistent with this model.

Among the GBS products whose expression is up-regulatedat a neutral pH is BibA, a putative virulence factor that con-tributes to phagocytic resistance and a candidate vaccine anti-gen (50). One consequence of the marked pH-dependent reg-ulation of bibA expression is that antibodies to BibA were fullyactive in supporting opsonophagocytic killing of GBS grown atpH 7.0 but were ineffective against organisms grown at pH 5.5.These results imply that pH-dependent regulation may be acritical factor in the effectiveness of vaccine-induced immunity.In the case of BibA, for example, it is likely that antibodieswould protect against bloodstream infection (neutral pH) butnot against vaginal colonization (acidic pH). In contrast, ex-pression of other potential vaccine antigens is upregulated in alow-pH environment. In particular, we observed increased ex-pression of pilus components at an acidic pH. Since pilusproteins have been implicated in epithelial adherence by GBS,increased pilus expression in the acidic milieu of the vaginamay enhance GBS colonization at this site (35, 46).

Taken together, the results of this study identify a centralrole of the CsrRS two-component system in the adaptation ofGBS to changes in environmental pH. One implication of thisregulatory response is that key virulence factors are up-regu-lated during the transition from the maternal birth canal tofetal tissues during peripartum infection. A further implicationof this work is that pH-dependent regulation of GBS surfaceproteins can affect the efficacy of immune responses directed

against such antigens, an important new insight for GBS vac-cine design strategies.

ACKNOWLEDGMENTS

This work was supported by internal funding from Novartis Vaccinesand by grant AI59502 from the National Institutes of Health (toM.R.W.). The funders had no role in the study design, data collectionand analysis, decision to publish, or preparation of the manuscript.

The authors do not have a commercial or other association thatmight pose a conflict of interest.

We are grateful to Immaculada Margarit Y Ros for kindly providing2603 V/R microarray slides. We thank Mattia Bosello and Scilla Buc-cato for technical assistance and Alfredo Pezzicoli for useful discus-sions.

REFERENCES

1. Ajdic, D., W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B.Carson, C. Primeaux, R. Tian, S. Kenton, H. Jia, S. Lin, Y. Qian, S. Li, H.Zhu, F. Najar, H. Lai, J. White, B. A. Roe, and J. J. Ferretti. 2002. Genomesequence of Streptococcus mutans UA159, a cariogenic dental pathogen.Proc. Natl. Acad. Sci. USA 99:14434–14439.

2. Bayles, K. W. 2007. The biological role of death and lysis in biofilm devel-opment. Nat. Rev. Microbiol. 5:721–726.

3. Bernish, B., and I. van de Rijn. 1999. Characterization of a two-componentsystem in Streptococcus pyogenes which is involved in regulation of hyaluronicacid production. J. Biol. Chem. 274:4786–4793.

4. Biswas, I., A. Gruss, S. D. Ehrlich, and E. Maguin. 1993. High-efficiencygene inactivation and replacement system for gram-positive bacteria. J. Bac-teriol. 175:3628–3635.

5. Boskey, E. R., K. M. Telsch, K. J. Whaley, T. R. Moench, and R. A. Cone.1999. Acid production by vaginal flora in vitro is consistent with the rate andextent of vaginal acidification. Infect. Immun. 67:5170–5175.

6. Botta, G. A. 1979. Hormonal and type-dependent adhesion of group Bstreptococci to human vaginal cells. Infect. Immun. 25:1084–1086.

7. Brodeur, B. R., M. Boyer, I. Charlebois, J. Hamel, F. Couture, C. R. Rioux,and D. Martin. 2000. Identification of group B streptococcal Sip protein,which elicits cross-protective immunity. Infect. Immun. 68:5610–5618.

8. Brunskill, E. W., and K. W. Bayles. 1996. Identification and molecularcharacterization of a putative regulatory locus that affects autolysis in Staph-ylococcus aureus. J. Bacteriol. 178:611–618.

9. Chong, P., L. Drake, and I. Biswas. 2008. Modulation of covR expression inStreptococcus mutans UA159. J. Bacteriol. 190:4478–4488.

10. Clancy, A., J. W. Loar, C. D. Speziali, M. Oberg, D. E. Heinrichs, and C. E.Rubens. 2006. Evidence for siderophore-dependent iron acquisition in groupB streptococcus. Mol. Microbiol. 59:707–721.

11. Cornacchione, P., L. Scaringi, K. Fettucciari, E. Rosati, R. Sabatini, G.Orefici, C. von Hunolstein, A. Modesti, A. Modica, F. Minelli, and P. Mar-coni. 1998. Group B streptococci persist inside macrophages. Immunology93:86–95.

12. Cotter, P. D., and C. Hill. 2003. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol. Mol. Biol. Rev. 67:429–453.

13. Cundell, D. R., B. J. Pearce, J. Sandros, A. M. Naughton, and H. R. Masure.1995. Peptide permeases from Streptococcus pneumoniae affect adherence toeucaryotic cells. Infect. Immun. 63:2493–2498.

14. Darmstadt, G. L., L. Mentele, A. Podbielski, and C. E. Rubens. 2000. Roleof group A streptococcal virulence factors in adherence to keratinocytes.Infect. Immun. 68:1215–1221.

15. Edwards, M. S., and C. J. Baker. 2005. Group B streptococcal infections inelderly adults. Clin. Infect. Dis. 41:839–847.

16. Federle, M. J., and J. R. Scott. 2002. Identification of binding sites for thegroup A streptococcal global regulator CovR. Mol. Microbiol. 43:1161–1172.

17. Froehlich, B. J., C. Bates, and J. R. Scott. 2009. Streptococcus pyogenesCovRS mediates growth in iron starvation and in the presence of the humancationic antimicrobial peptide LL-37. J. Bacteriol. 191:673–677.

18. Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Stur-devant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott, and J. M.Musser. 2002. Virulence control in group A Streptococcus by a two-compo-nent gene regulatory system: global expression profiling and in vivo infectionmodeling. Proc. Natl. Acad. Sci. USA 99:13855–13860.

19. Gruening, P., M. Fulde, P. Valentin-Weigand, and R. Goethe. 2006. Struc-ture, regulation, and putative function of the arginine deiminase system ofStreptococcus suis. J. Bacteriol. 188:361–369.

20. Gryllos, I., R. Grifantini, A. Colaprico, S. Jiang, E. Deforce, A. Hakansson,J. L. Telford, G. Grandi, and M. R. Wessels. 2007. Mg2� signalling definesthe group A streptococcal CsrRS (CovRS) regulon. Mol. Microbiol. 65:671–683.

21. Gryllos, I., J. C. Levin, and M. R. Wessels. 2003. The CsrR/CsrS two-component system of group A Streptococcus responds to environmentalMg2�. Proc. Natl. Acad. Sci. USA 100:4227–4232.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

22. Gryllos, I., H. J. Tran-Winkler, M. F. Cheng, H. Chung, R. Bolcome III, W.Lu, R. I. Lehrer, and M. R. Wessels. 2008. Induction of group A Streptococ-cus virulence by a human antimicrobial peptide. Proc. Natl. Acad. Sci. USA105:16755–16760.

23. Hansen, S. M., N. Uldbjerg, M. Kilian, and U. B. Sorensen. 2004. Dynamicsof Streptococcus agalactiae colonization in women during and after preg-nancy and in their infants. J. Clin. Microbiol. 42:83–89.

24. Jan, G., P. Leverrier, V. Pichereau, and P. Boyaval. 2001. Changes in proteinsynthesis and morphology during acid adaptation of Propionibacteriumfreudenreichii. Appl. Environ. Microbiol. 67:2029–2036.

25. Janulczyk, R., S. Ricci, and L. Bjorck. 2003. MtsABC is important formanganese and iron transport, oxidative stress resistance, and virulence ofStreptococcus pyogenes. Infect. Immun. 71:2656–2664.

26. Jayaraman, G. C., J. E. Penders, and R. A. Burne. 1997. Transcriptionalanalysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regu-lation of expression in response to heat shock and environmental acidifica-tion. Mol. Microbiol. 25:329–341.

27. Jiang, S. M., M. J. Cieslewicz, D. L. Kasper, and M. R. Wessels. 2005.Regulation of virulence by a two-component system in group B streptococ-cus. J. Bacteriol. 187:1105–1113.

28. Jiang, S. M., N. Ishmael, J. D. Hotopp, M. Puliti, L. Tissi, N. Kumar, M. J.Cieslewicz, H. Tettelin, and M. R. Wessels. 2008. Variation in the group BStreptococcus CsrRS regulon and effects on pathogenicity. J. Bacteriol. 190:1956–1965.

29. Kappes, R. M., B. Kempf, and E. Bremer. 1996. Three transport systems forthe osmoprotectant glycine betaine operate in Bacillus subtilis: characteriza-tion of OpuD. J. Bacteriol. 178:5071–5079.

30. Kubín, V., Z. Jiraskova, and J. Franek. 1983. The effect of pH on theadherence of group B streptococci to epithelial cells. Folia Microbiol.(Praha) 28:62–64.

31. Lamy, M. C., M. Zouine, J. Fert, M. Vergassola, E. Couve, E. Pellegrini, P.Glaser, F. Kunst, T. Msadek, P. Trieu-Cuot, and C. Poyart. 2004. CovS/CovR of group B streptococcus: a two-component global regulatory systeminvolved in virulence. Mol. Microbiol. 54:1250–1268.

32. Lauer, P., C. D. Rinaudo, M. Soriani, I. Margarit, D. Maione, R. Rosini,A. R. Taddei, M. Mora, R. Rappuoli, G. Grandi, and J. L. Telford. 2005.Genome analysis reveals pili in group B Streptococcus. Science 309:105.

33. Levin, J. C., and M. R. Wessels. 1998. Identification of csrR/csrS, a geneticlocus that regulates hyaluronic acid capsule synthesis in group A Streptococ-cus. Mol. Microbiol. 30:209–219.

34. Maione, D., I. Margarit, C. D. Rinaudo, V. Masignani, M. Mora, M.Scarselli, H. Tettelin, C. Brettoni, E. T. Iacobini, R. Rosini, N. D’Agostino,L. Miorin, S. Buccato, M. Mariani, G. Galli, R. Nogarotto, V. N. Dei, F.Vegni, C. Fraser, G. Mancuso, G. Teti, L. C. Madoff, L. C. Paoletti, R.Rappuoli, D. L. Kasper, J. L. Telford, and G. Grandi. 2005. Identification ofa universal group B Streptococcus vaccine by multiple genome screen. Sci-ence 309:148–150.

35. Maisey, H. C., M. Hensler, V. Nizet, and K. S. Doran. 2007. Group Bstreptococcal pilus proteins contribute to adherence to and invasion of brainmicrovascular endothelial cells. J. Bacteriol. 189:1464–1467.

36. Marques, M. B., D. L. Kasper, M. K. Pangburn, and M. R. Wessels. 1992.Prevention of C3 deposition by capsular polysaccharide is a virulence mech-anism of type III group B streptococci. Infect. Immun. 60:3986–3993.

37. Mereghetti, L., I. Sitkiewicz, N. M. Green, and J. M. Musser. 2008. Extensiveadaptive changes occur in the transcriptome of Streptococcus agalactiae(group B streptococcus) in response to incubation with human blood. PLoSONE 3:e3143.

38. Mereghetti, L., I. Sitkiewicz, N. M. Green, and J. M. Musser. 2008. Remod-eling of the Streptococcus agalactiae transcriptome in response to growthtemperature. PLoS ONE 3:e2785.

39. Miller, A. A., N. C. Engleberg, and V. J. DiRita. 2001. Repression of viru-lence genes by phosphorylation-dependent oligomerization of CsrR at targetpromoters in S. pyogenes. Mol. Microbiol. 40:976–990.

40. Miller, J. H. 1992. A short course in bacterial genetics: a laboratory manualand handbook for Escherichia coli and related bacteria. Cold Spring HarborLaboratory Press, Plainview, NY.

41. Obis, D., A. Guillot, J. C. Gripon, P. Renault, A. Bolotin, and M. Y. Mistou.1999. Genetic and biochemical characterization of a high-affinity betaineuptake system (BusA) in Lactococcus lactis reveals a new functional organi-zation within bacterial ABC transporters. J. Bacteriol. 181:6238–6246.

42. Paavonen, J. 1983. Physiology and ecology of the vagina. Scand. J. Infect.Dis. Suppl. 40:31–35.

43. Papp-Wallace, K. M., and M. E. Maguire. 2006. Manganese transport andthe role of manganese in virulence. Annu. Rev. Microbiol. 60:187–209.

44. Patton, T. G., S. J. Yang, and K. W. Bayles. 2006. The role of proton motiveforce in expression of the Staphylococcus aureus cid and lrg operons. Mol.Microbiol. 59:1395–1404.

45. Perez-Casal, J., J. A. Price, E. Maguin, and J. R. Scott. 1993. An M proteinwith a single C repeat prevents phagocytosis of Streptococcus pyogenes: use ofa temperature-sensitive shuttle vector to deliver homologous sequences tothe chromosome of S. pyogenes. Mol. Microbiol. 8:809–819.

46. Pezzicoli, A., I. Santi, P. Lauer, R. Rosini, D. Rinaudo, G. Grandi, J. L.Telford, and M. Soriani. 2008. Pilus backbone contributes to group B Strep-tococcus paracellular translocation through epithelial cells. J. Infect. Dis.198:890–898.

47. Poyart, C., E. Pellegrini, O. Gaillot, C. Boumaila, M. Baptista, and P.Trieu-Cuot. 2001. Contribution of Mn-cofactored superoxide dismutase(SodA) to the virulence of Streptococcus agalactiae. Infect. Immun. 69:5098–5106.

48. Poyart, C., and P. Trieu-Cuot. 1997. A broad-host-range mobilizable shuttlevector for the construction of transcriptional fusions to beta-galactosidase ingram-positive bacteria. FEMS Microbiol. Lett. 156:193–198.

49. Samen, U., B. Gottschalk, B. J. Eikmanns, and D. J. Reinscheid. 2004.Relevance of peptide uptake systems to the physiology and virulence ofStreptococcus agalactiae. J. Bacteriol. 186:1398–1408.

49a.Santi, I., D. Maione, C. L. Galeotti, G. Grandi, J. L. Telford, and M. Soriani.2009. BibA induces opsonizing antibodies conferring in vivo protectionagainst Group B Streptococcus. J. Infect. Dis., in press.

50. Santi, I., M. Scarselli, M. Mariani, A. Pezzicoli, V. Masignani, A. Taddei, G.Grandi, J. L. Telford, and M. Soriani. 2007. BibA: a novel immunogenicbacterial adhesin contributing to group B Streptococcus survival in humanblood. Mol. Microbiol. 63:754–767.

51. Scarpelli, E. M. 1978. Intrapulmonary foam at birth: an adaptational phe-nomenon. Pediatr. Res. 12:1070–1076.

52. Schrag, S. J., and B. J. Stoll. 2006. Early-onset neonatal sepsis in the era ofwidespread intrapartum chemoprophylaxis. Pediatr. Infect. Dis. J. 25:939–940.

53. Sperandio, B., C. Gautier, S. McGovern, D. S. Ehrlich, P. Renault, I. Mar-tin-Verstraete, and E. Guedon. 2007. Control of methionine synthesis anduptake by MetR and homocysteine in Streptococcus mutans. J. Bacteriol.189:7032–7044.

54. Takahashi, S., Y. Nagano, N. Nagano, O. Hayashi, F. Taguchi, and Y.Okuwaki. 1995. Role of C5a-ase in group B streptococcal resistance toopsonophagocytic killing. Infect. Immun. 63:4764–4769.

55. Tamura, G. S., A. Nittayajarn, and D. L. Schoentag. 2002. A glutaminetransport gene, glnQ, is required for fibronectin adherence and virulence ofgroup B streptococci. Infect. Immun. 70:2877–2885.

56. Tettelin, H., V. Masignani, M. J. Cieslewicz, J. A. Eisen, S. Peterson, M. R.Wessels, I. T. Paulsen, K. E. Nelson, I. Margarit, T. D. Read, L. C. Madoff,A. M. Wolf, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. T. DeBoy, A. S.Durkin, J. F. Kolonay, R. Madupu, M. R. Lewis, D. Radune, N. B. Fedorova,D. Scanlan, H. Khouri, S. Mulligan, H. A. Carty, R. T. Cline, S. E. Van Aken,J. Gill, M. Scarselli, M. Mora, E. T. Iacobini, C. Brettoni, G. Galli, M.Mariani, F. Vegni, D. Maione, D. Rinaudo, R. Rappuoli, J. L. Telford, D. L.Kasper, G. Grandi, and C. M. Fraser. 2002. Complete genome sequence andcomparative genomic analysis of an emerging human pathogen, serotype VStreptococcus agalactiae. Proc. Natl. Acad. Sci. USA 99:12391–12396.

57. Ware, D., Y. Jiang, W. Lin, and E. Swiatlo. 2006. Involvement of potD inStreptococcus pneumoniae polyamine transport and pathogenesis. Infect. Im-mun. 74:352–361.

58. Warren, M. A., L. M. Kucharski, A. Veenstra, L. Shi, P. F. Grulich, andM. E. Maguire. 2004. The CorA Mg2� transporter is a homotetramer. J.Bacteriol. 186:4605–4612.

59. Weinrick, B., P. M. Dunman, F. McAleese, E. Murphy, S. J. Projan, Y. Fang,and R. P. Novick. 2004. Effect of mild acid on gene expression in Staphylo-coccus aureus. J. Bacteriol. 186:8407–8423.

60. Wessels, M. R., L. C. Paoletti, A. K. Rodewald, F. Michon, J. DiFabio, H. J.Jennings, and D. L. Kasper. 1993. Stimulation of protective antibodiesagainst type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine. Infect. Immun. 61:4760–4766.

61. Yamamoto, Y., C. Poyart, P. Trieu-Cuot, G. Lamberet, A. Gruss, and P.Gaudu. 2005. Respiration metabolism of group B Streptococcus is activatedby environmental haem and quinone and contributes to virulence. Mol.Microbiol. 56:525–534.

62. Zawaneh, S. M., E. M. Ayoub, H. Baer, A. C. Cruz, and W. N. Spellacy. 1979.Factors influencing adherence of group B streptococci to human vaginalepithelial cells. Infect. Immun. 26:441–447.



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

csrrs regulates group b streptococcus virulence gene … · 2017. 8. 15. · cate that the csrrs...

Documents