The Sensor Histidine Kinase RgfC Affects Group B StreptococcalVirulence Factor Expression Independent of Its Response RegulatorRgfA

Claire Gendrin,a Annalisa Lembo,a* Christopher Whidbey,a,b Kellie Burnside,a* Jessica Berry,a Lisa Ngo,a Anirban Banerjee,c*Liang Xue,d Justine Arrington,d Kelly S. Doran,c W. Andy Tao,d Lakshmi Rajagopala,b

Department of Pediatric Infectious Diseases, University of Washington School of Medicine and Seattle Children’s Research Institute, Seattle, Washington, USAa;Department of Global Health, University of Washington, Seattle, Washington, USAb; Department of Biology and Center for Microbial Sciences, San Diego State University,San Diego, California, USAc; Department of Biochemistry, Purdue University, West Lafayette, Indiana, USAd

Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptom-atic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and incertain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number oftwo-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC andthe response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certainGBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfAprotein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that de-letion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutantincreased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopep-tides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine resi-dues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator.Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expressioncompared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associatedwith GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expressionof virulence factors and GBS pathogenesis.

All living organisms sense and adapt to dynamic changes intheir environment using signaling systems. Signaling in pro-

karyotic organisms is achieved primarily by two-component sig-naling systems (TCS) that regulate gene expression in response toenvironmental signals (1–5). A typical two-component systemcomprises a membrane-associated sensor histidine kinase that re-sponds to an environmental signal and phosphorylates its cognateDNA binding response regulator at an aspartate residue. Phos-phorylation often alters the affinity of the response regulator to itstarget promoters, regulating gene expression.

Group B streptococci (GBS) or Streptococcus agalactiae strainsare a common cause of bacterial infections in human newbornsand are emerging pathogens of adult humans (6). These bacteriareside as commensal organisms in the lower gastrointestinal andgenital tracts of healthy adult women. Human neonates are atrisk for GBS infections due to ascending infection from thelower genital tract or from aspiration of contaminated amni-otic/vaginal fluids during birth. GBS can disseminate into mul-tiple neonatal organs, causing pneumonia, sepsis, and menin-gitis (for reviews, see references 6–8). Thus, GBS has toefficiently adapt to the changing environmental conditions en-countered during its life cycle.

Signaling mechanisms that enable GBS transition from com-mensal environments to invasive niches is not understood (8).Like other prokaryotic organisms, signaling in GBS is achievedprimarily by two-component signaling systems (TCS) that regu-late gene expression in response to external signals (1, 3, 4). TheGBS genome sequence indicates the presence of 18 to 20 TCS (9),

and the roles of a few TCS have been well characterized and in-clude CovR/CovS (10–15), DltR/DltS (16), CiaH/CiaR (17),SAK_0188/0189 (18), RgfC/A (19, 20), and FpsR/S (21). Previousstudies have indicated that GBS deficient for RgfC/A exhibit re-duced adherence to extracellular matrix components due to al-tered expression of the fibrinogen binding proteins, such as FbsA

Received 3 October 2014 Returned for modification 26 October 2014Accepted 28 December 2014

Accepted manuscript posted online 5 January 2015

Citation Gendrin C, Lembo A, Whidbey C, Burnside K, Berry J, Ngo L, Banerjee A,Xue L, Arrington J, Doran KS, Tao WA, Rajagopal L. 2015. The sensor histidinekinase RgfC affects group B streptococcal virulence factor expressionindependent of its response regulator RgfA. Infect Immun 83:1078 –1088.doi:10.1128/IAI.02738-14.

Editor: A. Camilli

Address correspondence to Lakshmi Rajagopal,lakshmi.rajagopal@seattlechildrens.org.

* Present address: Annalisa Lembo, Bellevue Community College, Bellevue,Washington, USA; Kellie Burnside, LaCorp Clinical Trials, Seattle, Washington, USA;Anirban Banerjee, Department of Biosciences & Bioengineering, IIT-Bombay,Mumbai, India.

C.G. and A.L. contributed equally.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02738-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02738-14

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and FbsB (19, 20). However, in certain GBS clinical isolates thatwere obtained from infected newborns (e.g., A909 and H36B), apoint mutation in rgfA results in the premature truncation of theresponse regulator (9; also see the schematic in Fig. 1A and B); thisfeature was observed in another 61 GBS strains (21). The trun-cated RgfA protein lacks the C-terminal DNA binding domainnecessary for promoter binding and regulation of gene expression.The presence of a spontaneous mutation resulting in prematuretruncation of the response regulator rgfA in certain GBS strainsand regulation of these genes by CovR/S (12) prompted us todetermine if RgfC function was abolished in strains lacking a func-tional RgfA. Here, we show that in strains lacking a functionalRgfA, deletion of RgfC increased the expression of virulence fac-tors and systemic GBS infection. These data suggest that nonspe-cific kinase activity in the absence of the cognate response regula-tor affects GBS virulence.

MATERIALS AND METHODSGeneral growth. The strains, plasmids, and primers used in this study arelisted in Table S1 in the supplemental material. The wild-type (WT) GBSstrains A909 and COH1 are clinical isolates belonging to capsular poly-saccharide serotype 1a (22) and serotype III (23), respectively. Routinecultures of GBS were performed in tryptic soy broth (TSB; Difco Labora-tories, Detroit, MI) in 5% CO2 at 37°C. Routine cultures of Escherichia coliwere performed in Luria-Bertani broth (LB; Difco Laboratories, Detroit,MI) at 37°C. All chemicals were purchased from Sigma-Aldrich (St. Louis,MO), unless mentioned otherwise. GBS cell growth was monitored at 600nm after incubation in 5% CO2 at 37°C unless mentioned otherwise.Antibiotics were added at the following concentrations when necessary:for GBS, erythromycin, 1 �g ml�1; kanamycin, 1,000 �g ml�1; chloram-phenicol, 2.5 to 5 �g ml�1; spectinomycin, 300 �g ml�1; for E. coli, eryth-romycin, 300 �g ml�1; kanamycin, 50 �g ml�1; chloramphenicol, 10 �gml�1; spectinomycin, 50 �g ml�1.

Construction of �rgfCA mutants. Approximately 1 kb of DNA lo-cated upstream of rgfA was amplified using the primers upstream F andupstream R and high-fidelity PCR (Invitrogen, CA, USA). Likewise, 1 kbof DNA downstream of rgfC was amplified using primers downstream Fand downstream R as described above. The gene conferring kanamycinresistance (�km-2) was amplified from pCIV2 (24) using primers KanFand KanR for allelic replacement of rgfC and rgfA (denoted rgfCAd forA909). Subsequently, splicing by overlap extension PCR (SOEing PCR)(25) was performed to introduce the antibiotic resistance gene betweenthe flanking regions of rgfC and rgfA described above. The PCR fragmentthen was ligated into the temperature-sensitive vector pHY304 (26), andthe resulting plasmid (�rgfCA::kn) was electroporated into GBS A909 andCOH1 as described previously (27). Selection and screening for the dou-ble crossover (�rgfCA::kn) was performed as described previously (27).PCR was used to verify the presence of �km-2 and the absence of rgfCA.The A909 �rgfCAd strain was used as the parent strain to derive the�rgfCAd �cspA double mutant. The gene cspA was allelically replaced withaad9, encoding spectinomycin resistance in pLZ12spec (28), using SOE-ing PCR and PCR products obtained from primer pairs cpsAupF andcspAupR, specF and specR, and cpsAdnF and cspAdnR. PCR was usedto verify the allelic replacement of cpsA with aad9 (encoding spectino-mycin resistance). The complementing pRgfC and pRgfAd plasmidswere constructed by amplifying rgfC using primers pDCSAK_1917F andpDCSAK_1917R and rgfAd using primers pDCSAK_1918F and pDCSAK_1918R, respectively, and A909 genomic DNA was used as the template.The PCR fragment was digested with restriction enzymes present onthe primer sequence(s) and then cloned into the multiple cloning siteof the GBS complementation vector pDC123 downstream of the con-stitutive promoter as described previously (27, 29). The complement-ing plasmid for corrected pRgfCA, i.e., functional rgfA without thepremature stop codon, was obtained by amplifying rgfCA with GBS

COH1 DNA as the template using the Gibson Assembly cloning kit byfollowing the manufacturer’s instructions (New England BioLabs,USA). Overlapping primers (RgfA-Gibson-F, RgfA-Gibson-FC, RgfC-Gibson-R, and RgfC-Gibson-RC) were designed to amplify rgfCAfrom COH1 and the GBS complementation vector pDC123 such thatthe insert DNA was downstream of the constitutive promoter inpDC123. In all cases, DNA sequencing was used to verify the orienta-tion and sequence of cloned plasmids. The complementing plasmidsthen were electroporated into the A909 �rgfCAd mutant using meth-ods described previously (27).

Virulence analysis. All animal experiments were approved by the In-stitutional Animal Care and Use Committee (protocol 13311) and per-formed using guidelines in the Guide for the Care and Use of LaboratoryAnimals (8th ed.) (30).

Neonatal rat sepsis model. Virulence analysis using the neonatal ratsepsis model of infection was performed as described previously (31).Briefly, time-mated, female Sprague Dawley rats were obtained fromCharles River Laboratories (MA, USA). WT A909 and �rgfCAd strainswere grown to an optical density at 600 nm (OD600) of 0.3, washed, re-suspended in phosphate-buffered saline, and used as the inoculum.Groups of six rat pups (24 to 48 h of age) were given 10-fold serial dilu-tions of each strain by intraperitoneal injection, and the pups werechecked for signs of morbidity every 8 h for 72 h. The 50% lethal dose(LD50; also called the 50% moribund dose [MD50]) estimates were de-rived using logistic regression models for the probability of death condi-tional on dose and strain as described previously (10). The experimentwas repeated twice, and P values of less than 0.05 were considered signif-icant.

GBS sepsis/meningitis model of infection. The murine model of he-matogenous GBS meningitis was used to compare the virulence potentialof strains used in this study (32, 33). Briefly, 6-week-old male CD-1 miceobtained from Charles River Laboratories (MA, USA) were injected viathe tail vein with 3 � 107 to 3 � 108 of either the WT A909 or isogenic�rgfCAd strain. Spleen and brains from infected mice were collected asep-tically 24 h postinfection. Bacterial counts in spleen and brain homoge-nates were determined by plating serial 10-fold dilutions on TSB agar.Interleukin-6 (IL-6), MIP-2 (macrophage inflammatory protein 2, alsoknown as CXCL2), and KC (keratinocyte-derived chemokine, also knownas CXCL1; IL-8 homologue) enzyme-linked immunosorbent assays (ELI-SAs) were performed on supernatants from spleen and brain homoge-nates using the DuoSet kit as described by the manufacturer (R&D Sys-tems, USA).

Adherence and invasion of hBMEC. The human brain microvascularendothelial cell (hBMEC) line, immortalized by transfection with the sim-ian virus 40 (SV40) large T antigen (34), was used in these studies. Prop-agation of hBMEC and GBS adherence, invasion, and intracellular sur-vival were performed as described previously (33). A 2-fold increase ordecrease in adherence or invasion compared to the isogenic WT A909 wasconsidered significant, as described previously (33).

Phosphopeptide enrichment and MS. Total protein was isolatedfrom the WT A909, the �rgfCAd mutant, and �rgfCAd/pRgfC comple-mented strain as described previously (35, 36). Briefly, GBS were grown toan OD600 of 0.6 in TSB. The bacteria were washed twice with 0.25� ice-cold buffer containing phosphatase inhibitors (20 mM Tris, pH 7.5, 10mM NaF, 10 mM Na pyrophosphate, and 50 mM �-glycerophosphate).The cells subsequently were resuspended in lysis buffer (50 mM Tris, pH7.5, with the phosphatase inhibitors described above at a concentration of5 mM) and disrupted using the FastPrep FP101 bead beater (Bio 101). Thelysates were treated with DNase I and ultracentrifuged to pellet unlysedcells and cell debris at 25,000 � g as described previously (35, 36).Thesupernatant from each sample was normalized to contain equal amountsof protein. The proteins then were denatured and reduced in 4% SDS with10 mM dithiothreitol (DTT) at 95°C for 5 min. Subsequently, the proteinsamples were added to centrifugal filter units with a 30-kDa molecularmass cutoff (Microcon-30; Millipore) and washed with 8 M urea in 50

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mM Tris-HCl (pH 7.5). Proteins were alkylated with 30 mM iodoacet-amide for 20 min at room temperature in the dark. The samples werewashed again with 8 M urea in Tris-HCl before the buffer was changed to50 mM ammonium bicarbonate. The samples were digested overnight at37°C using sequencing-grade trypsin (1:50, trypsin to total protein). Thenext day, peptides were eluted from the filters using 50 mM ammoniumbicarbonate followed by 0.5 M NaCl. The peptide samples were desaltedusing Sep-Pak C18 columns according to the manufacturer’s instructions(Waters, USA) and dried using a Speedvac. From the peptide samples ofeach strain (800 �g), phosphopeptides were captured using a solublenanopolymer functionalized with titanium, PolyMAC-Ti (Tymora Ana-lytical, West Lafayette, IN), as described previously (37). Unbound non-phosphopeptides were washed away, and phosphopeptides were eluted asdescribed previously (37). The samples were analyzed by capillary liquidchromatography-nanoelectrospray tandem mass spectrometry (�LC-na-noESI-MS/MS) with a high-resolution hybrid linear ion trap orbitrap(LTQ-orbitrap Velos; Thermo Fisher) coupled to an Eksigent NanoLCUltra two-dimensional high-performance liquid chromatography (2DHPLC) system using methods described previously (36, 38). Data weresearched using Proteome Discoverer software with the SEQUEST algo-rithm using a 10-ppm precursor mass tolerance and a 0.6-Da MS/MSmass tolerance. The searches included variable modifications on cysteineresidues (�57.021 or �79.966), methionine residues (�15.995), and as-partate, serine, threonine, and tyrosine residues (�79.966) to identifyphosphorylation as described previously (35, 36, 38). Spectra weresearched against the GBS A909 genome database (accession numberNC_007432) with a 1% false discovery rate (FDR) based on a reversedecoy database search. Proteome Discoverer generated a reverse decoydatabase from the GBS A909 protein database, and any peptides passingthe initial filtering parameters of this decoy database were defined as falsepositives. Based on the number of random false positives matched to thedecoy database, Proteome Discoverer adjusted the minimum cross-cor-relation factor (Xcorr) filter for each charge state to meet the predeter-mined target FDR of 1%. Thus, each data set had its own passing param-eters. Phosphorylation site localization was determined from tandemmass spectra (39) with phosphoRS (40). The unique phosphopeptidesand nonphosphopeptides identified then were manually counted andcompared. For phosphopeptides with potentially ambiguous phosphory-lation, only the top-scored phosphorylation site was used for further anal-ysis.

Isolation and purification of GBS RNA. Total RNA from GBS wasisolated using the RNeasy minikit (Qiagen, Inc., Valencia, CA) as de-scribed previously (13). In brief, GBS strains were grown to an OD600 of0.6, centrifuged, washed in 1:1 mixed RNA Protect (Qiagen, Inc., Valen-cia, CA) and Tris-EDTA buffer, and resuspended in kit-supplied RLTbuffer. The cell suspensions were lysed through the use of a FastPrepFP101 bead beater (Bio 101), followed by clarification of the lysates viacentrifugation. The supernatants then were purified and DNase digested(Qiagen, Inc., Valencia, CA) as described by the RNeasy minikit manu-facturer’s instructions. RNA integrity and concentration were determinedusing an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA) or a Nano-Drop 1000 (NanoDrop, Wilmington, DE) for use in microarrays or quan-titative reverse transcription-PCR (qRT-PCR), respectively.

GBS microarrays and qRT-PCR. RNA was isolated from two inde-pendent biological replicates for each strain and purified as describedabove. Purified RNA was sent to NimbleGen Systems, Inc. (Madison,WI), for full expression services. NimbleGen performed cDNA synthesis,labeling of the cDNA, and hybridization of the labeled cDNA to the Strep-tococcus agalactiae A909 chip (A4327-00-01; NimbleGen Systems, Inc.Madison, WI) according to company protocols. The chips were com-posed of 18 probes per target sequence, and each probe was replicated fivetimes (see www.nimblegen.com/products/exp/index.html for details).Microarray data were interpreted and analyzed using the program Gene-Spring GX (version 7.3.1; Agilent Technologies, Santa Clara, CA). Geneswith statistically significant differences among groups were calculated us-

ing the Welch t test (parametric, with variances not assumed equal) with aP value cutoff of 0.05 and an associated Benjamini and Hochberg FDRmultiple-testing correction (about 5.0% of the identified genes would beexpected to pass the restriction by chance) (41). Standard error propaga-tion was calculated using the Delta method for ratios of means from thethree independent biological replicates for A909 (WT) and �rgfCAd

strains. All fold changes were defined as relative to the level for A909 WT.qRT-PCR was performed using a one-step QuantiTect SYBR green RT-PCR kit (Qiagen, Inc., Valencia, CA) as described previously (13). Thereference gene used for all runs was the housekeeping ribosomal proteinS12 gene rpsL (10).

Sialic acid quantification of capsular polysaccharide (CPS). As ameasure of the amount of capsule produced by isogenic GBS strains,capsular sialic acid was quantified after acid hydrolysis of equivalentnumbers of whole cells and measured by HPLC as described previously(42). Briefly, equivalent numbers of whole cells of each GBS strainwere treated with 4N acetic acid for 1 h, and the sialic acid that wasrecovered was derivatized with fluorescent 1,2-diamino-4,5-methyl-ene-dioxybenzene (DMB) and quantified compared to a standardcurve using pure sialic acid (Sigma) similarly derivatized with DMB asdescribed previously (42).

Statistical analysis. Unless mentioned otherwise, the Mann-Whitneytest or unpaired t test was used to estimate differences between GBSstrains. These tests were performed using GraphPad Prism (version 5.0 forWindows; GraphPad Software, San Diego, CA, USA).

Microarray data accession number. The entire set of microarray datahas been deposited at the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE21563.

RESULTSGBS deficient in RgfC expression exhibit increased virulence.Our previous studies indicated that the CovR/S two-componentsystem regulated the expression of rgfC and rgfA in GBS serotypeIa, strain A909 (12). The GBS strain A909 is a clinical isolate ob-tained from an infected newborn (23, 43), and genome sequenc-ing indicated the presence of a point mutation encoding a stopcodon in rgfA that results in premature truncation of the responseregulator (9) (Fig. 1A and B). The same point mutation in rgfAalso was identified in other GBS clinical strains, such as H36B (9)(Fig. 1B) and 61 other GBS strains (21). To determine if RgfC hada significant role in gene regulation and virulence of GBS in strainslacking a functional RgfA, we constructed a strain deficient in rgfCexpression from the wild-type (WT) strain A909. Briefly, the en-tire coding sequence of rgfC and the nonfunctional rgfA was re-placed with a gene that conferred kanamycin resistance (here re-ferred to as �rgfCAd; see Materials and Methods). The growthcharacteristics of the �rgfCAd mutant were similar to those of WTA909 (see Fig. S1 in the supplemental material). We next com-pared the virulence potential of the �rgfCAd mutant to that of WTA909 using the neonatal rat sepsis model of infection describedpreviously (27, 31, 35). Interestingly, the 50% moribund dose(MD50; previously known as LD50) estimates of the �rgfCAd mu-tant were 10-fold lower than those for WT A909 (Table 1) (P 0.012). These results suggest that virulence of the �rgfCAd mutantis greater than that of WT A909.

We also examined the ability of the �rgfCAd mutant to cause in-fections in the adult murine model of GBS sepsis and meningitis (32).As the �rgfCAd strain demonstrated an increased ability to causebloodstream infections in neonatal rats, we hypothesized that thisstrain is more proficient than the WT for infections in adult mice. Totest this hypothesis, 3 � 108 CFU of either WT A909 or the �rgfCAd

mutant were injected into the tail vein of adult CD-1 mice (n 13),

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and the mice were monitored up to 15 days for signs of infection asdescribed previously (12, 32). Interestingly, we observed that all miceinfected with the �rgfCAd mutant succumbed to the infection within4 days postinfection, in contrast to the WT GBS strain A909, whereinonly 1 of 13 inoculated mice succumbed to the infection (Fig. 2). Thesurvival of mice infected with the rgfCAd-deficient strain was statisti-cally significant compared to that of mice infected with the WT (P

TABLE 1 Increased virulence of RgfC mutant in the neonatal rat modelof GBS sepsisa

Strain MD50 (CFU) 95% CI P value

WT (A909) 1.05 � 106 4.4 � 105–2.57 � 106 NA�rgfCAd 1.05 � 105 2.98 � 104–3.69 � 105 0.012a MD50 estimates and confidence intervals (CI) were calculated as described inMaterials and Methods. P values reflect the comparison of the value of the mutantstrain to that of WT A909. NA, not applicable.

FIG 1 Sequence alignment and open reading frame of the TCS RgfC/A in GBS. (A) rgfC encodes a sensor histidine kinase, rgfB shows homology to nucleases, andSAK_1920 encodes a putative glucose-specific phosphotransferase system (PTS) transporter. In GBS A909, RgfA is truncated by the nonsense mutation C342T;thus, it lacks the DNA binding domain. (B) Alignment of the available RgfA sequences shows that the nonsense mutation present in A909 also is found in H36B.

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0.0001). Together, these results indicated that the RgfC mutant ex-hibits increased virulence.

GBS deficient in RgfC exhibit increased dissemination. Wethen compared bacterial dissemination and blood-brain barrierpenetration of the �rgfCAd mutant to that of WT A909 in vivo. Tothis end, CD-1 mice (n 5) were infected with 3 � 107 CFU of theWT or the �rgfCAd mutant. At 24 h postinfection, mouse brainsand spleen were harvested and the number of CFU were enumer-ated as described previously (12, 32). These studies indicated thatthe average number of CFU of the �rgfCAd mutant obtained fromthe spleen and brain of the infected mice were significantly higherthan that of WT A909 (Fig. 3A and B) (P 0.007). We also com-pared the expression of cytokines, including KC (the murine func-tional homologue of IL-8), IL-6, and chemokines, such as CXCL2/MIP2, in the spleen and brain of mice infected with the WT or the�rgfCAd mutant. The results shown in Fig. 4A to C indicate asignificant increase in expression of KC, IL-6, and MIP-2 in spleens ofmice infected with the �rgfCAd mutant compared to expression inWT A909. Likewise, an increase in KC, IL-6, and MIP-2 expressionalso was observed in the brains of mice infected with the �rgfCAd

mutant (Fig. 4D to F). Taken together, these data indicate that theabsence of rgfC accelerated GBS systemic infections.

RgfC regulation of GBS gene expression. To determine if theabsence of RgfC affected gene expression in GBS A909, we per-formed microarray analysis using methods described previously(12, 35; also see Materials and Methods). The results shown inTable S2 in the supplemental material indicate that 101 genesshowed increased expression, and 115 (excluding rgfC-rgfA) genesshowed decreased expression in the A909 �rgfCAd mutant. Thisincluded an approximately 6-fold increase in expression of thesurface-associated serine protease known as CspA that was previ-ously described to promote GBS resistance to opsonophagocytickilling (44, 45) (see Table S2). Furthermore, increased expressionof genes encoding the sialic acid-rich capsular polysaccharide(Sia-CPS) critical for GBS virulence (46, 47) also was observed.Other genes that showed increased expression in the �rgfCAd

strain include genes predicted to be important for metabolicfunctions (see Table S2). We observed a significant increase ingene expression of metabolite transporters, such as a hydro-phobic amino acid uptake system (18- to 50-fold; SAK_1594 toSAK_1597), a branched-chain amino acid uptake system (8.5-

fold; SAK_1575), and components of the phosphotransferase sys-tem (2.5 to 7-fold; SAK_0399, SAK_1909, SAK_0398, SAK_0400,SAK_0523, SAK_ 0529, SAK_0915, SAK_0530, SAK_0257, andSAK_1920). Increased expression of such genes may facilitate GBSsurvival and nutrient acquisition during infection and may in partaccount for the increased CFU observed during systemic infec-tions (Fig. 3). A large number of genes that showed decreasedexpression in the �rgfCAd mutant are proteins of unknown func-tion (see Table S2). Downregulated genes included rgfC-rgfA(SAK_1917-SAK_191718), which served as controls in these anal-yses. Previous studies on RgfC/A indicated that this TCS regulatedthe expression of genes encoding fibrinogen binding proteins suchas FbsA and FbsB in GBS encoding a functional RgfA (19). Themicroarray analysis revealed that expression of fbsA and fbsB inthe A909 �rgfCAd mutant was not significantly different from thatof WT A909 and is consistent with observations that adherenceand invasion of the A909 �rgfCAd mutant to hBMEC was similarto that of WT A909 (see Fig. S2). qRT-PCR analysis confirmedthat the expression of genes associated with GBS pathogenesis, i.e.,cspA and Sia-CPS genes, such as cpsH, cpsK, neuB, and SAK_1593,was increased in the A909 �rgfCAd mutant (Table 2). Comple-mentation of the A909 �rgfCAd mutant with a plasmid encodingonly RgfC restored transcription of these genes to WT levels (Ta-ble 2), whereas complementation with either a plasmid encodingRgfAd or both RgfC and a functional RgfA did not (Table 2). Wefurther observed that the deletion of rgfC-rgfA in GBS encoding a

FIG 2 �rgfCAd mutant shows increased virulence in the adult mouse model ofGBS infection. Thirteen 6-week-old male CD-1 mice were intravenously in-jected with 3 � 108 CFU of either the WT or �rgfCAd strain. A Kaplan-Meiersurvival curve shows the percent survival of mice after the infection. Note thatall of the mice infected with the �rgfCAd mutant succumbed to the infectionwithin 4 days postinoculation, in contrast to the WT (P 0.0001 by log-ranktest).

FIG 3 Increased infection in the spleen and brain of mice infected with the�rgfCAd mutant. Five 6-week-old male CD-1 mice were intravenously injectedwith 3 � 107 CFU of the WT or �rgfCAd strain. At approximately 24 h postin-fection, brains and spleens were harvested from the infected mice and CFUwere enumerated. Note that mice infected with the GBS �rgfCAd mutant haveincreased CFU in the brains and spleens (**, P 0.007 by Mann-Whitneytest).

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functional RgfA, such as the serotype III strain COH1, did notresult in increased virulence or increased expression of cspA andcapsular polysaccharide genes compared to the isogenic WTCOH1 (see Fig. S3). Collectively, these data suggest that RgfC canhave nonspecific activity in the absence of functional RgfA.

Sia-CPS levels in the A909 �rgfCAd mutant. Sialylation of theGBS capsular polysaccharide is critical for GBS bloodstream in-fections and pathogenesis (46, 48). To determine if the increase ingene expression of the capsular polysaccharide operon correlatedwith increased sialic acid levels in the A909 �rgfCAd mutant, wecompared sialic acid levels in cell surface-associated capsular poly-saccharide using HPLC as described previously (42, 49). The re-sults shown in Fig. 5A indicate that sialic acid levels were higher inthe �rgfCAd mutant than in WT A909 (P 0.01), which could in

part contribute to the enhanced virulence of the �rgfCAd mutant.Increased sialic acid levels were not observed in the COH1 �rgfCAmutant (see Fig. S3 in the supplemental material).

Role of CspA in virulence potential of �rgfCAd mutant. Weexamined whether the increase in expression of CspA contributedto the increase in virulence potential of the rgfCAd mutant. To testthis possibility, we constructed a double mutant that was deficientfor both CspA and rgfCAd in GBS A909 (a �rgfCAd �cspA strain;see Materials and Methods). Subsequently, the virulence potentialof the �rgfCAd �cspA double mutant was compared to those of theWT and �rgfCAd strains using the adult murine model of GBSinfection described previously. The results shown in Fig. 5B indi-cate that the increase in virulence of the rgfCAd mutant was dimin-ished in the �rgfCAd �cspA double mutant (P 0.004). These

FIG 4 Increased chemokine and cytokine expression in spleens of mice infected with the �rgfCAd mutant. Five 6-week-old male CD-1 mice were intravenouslyinjected with 3 � 107 CFU of the WT or �rgfCAd strain. At approximately 24 h postinfection, spleens and brains were harvested from the infected mice, and theexpression of KC (IL-8 equivalent), IL-6, and CXCL2/MIP2 was measured in the homogenates. Note that mice infected with the GBS �rgfCAd mutant haveincreased levels of KC, CXCL2/MIP-2, and IL-6 (*, P 0.01; **, P 0.007 by Mann-Whitney test).

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data indicate that the increase in CspA expression contributes tothe increase in virulence potential of the RgfC mutant.

RgfC regulation of protein phosphorylation. To determine ifthe absence of RgfC altered protein phosphorylation, we per-formed phosphopeptide enrichment analysis to identify peptides/proteins that were differentially phosphorylated between WTA909, the rgfCAd mutant, and the complemented strain carryingpRgfC (see Materials and Methods). Although the phosphopro-teomic analysis that we utilized is optimized to enrich and identifyS/T/Y phosphorylation, we minimized acid treatment to alsoidentify potential D/H phosphosites. The results obtained fromthese analyses are shown in Table 3. Table 3 indicates aspartatephosphopeptides that were identified only in RgfC-expressingstrains and correspond to a transcriptional regulator and the ty-rosine kinase CpsD (Fig. 6 shows a sample peptide sequence/spec-trum). These results suggest that RgfC directly or indirectly regu-lates phosphorylation of these peptides/proteins. Also, it is worthnoting that two aspartate phosphopeptides corresponding to amethyltransferase were identified in the RgfCAd mutant (Table 3),suggesting that RgfC influences dephosphorylation of the meth-yltransferase in GBS A909. Interestingly, we also observed changesin C/S/T/Y phosphorylation between RgfC-proficient and -defi-cient strains. This included 3 phosphopeptides in RgfC-proficientstrains and 12 in the RgfC-deficient strain (Table 3). Further stud-ies are essential to determine how these phosphorylation/dephos-phorylation events regulate protein function and for a better un-derstanding of the role of RgfC in GBS.

Collectively, our studies indicate that RgfC functions as a non-specific or orphan kinase in GBS strains with a mutation in RgfA,and this has an impact on GBS virulence. A detailed understand-ing of two-component regulation is necessary for the evaluation ofeither histidine kinases or cognate response regulators as an anti-microbial target(s) in strategies to prevent bacterial infections inhumans.

DISCUSSION

Signaling systems are critical for bacterial adaptation to changingenvironmental conditions. As GBS encounters diverse niches inits life cycle, it is likely that the approximately 20 TCS encoded byGBS sense diverse environmental signals and contribute to bacte-rial environmental adaptation. However, our understanding ofenvironmental signals that are sensed by these various TCS andmechanisms of gene regulation is incomplete. The sensor histi-dine kinase/response regulator RgfC/A initially was identified asregulating the expression of genes such as fbsA and fbsB, which areimportant for GBS binding to extracellular matrix componentssuch as fibrinogen (19, 20). However, the presence of a spontane-

ous mutation resulting in premature truncation of the responseregulator rgfA in certain GBS strains and regulation of these genesby CovR/S (12) prompted us to determine if RgfC function wasabolished in strains lacking a functional RgfA. We provide evi-dence that deletion of rgfC in strains encoding a nonfunctionalRgfA resulted in altered expression of �200 genes and includedcritical virulence components, such as the serine protease CspAand the sialic acid capsular polysaccharide. Control of these genessuggests that RgfC regulation of gene expression is altered in theabsence of a functional RgfA and that sensor kinase has a broaderrole in GBS virulence than previously appreciated. Support for

TABLE 2 RgfC regulates expression of capsular polysaccharide and serine protease genes in GBS A909

Locus

Relative gene expressiona

�909 �rgfCAd �909 �rgfCAd/pRgfC �909 �rgfCAd/pRgfAd �909 �rgfCAd/pRgfCAb

cspA 7.28 1.1 0.7 0.2 6.06 1.03 10.37 4.4cpsH 10.14 2.05 0.5 0.1 10.8 1.7 14.75 5.3cpsK 8.2 3.28 1.0 0.1 9.02 1.2 13.37 3.2neuB 9.65 1.01 1.36 0.24 8.7 2.45 9.6 3.3SAK_1593 25.49 3.08 0.74 .24 30 4.48 20.5 4.14a qRT-PCR was performed as described in Materials and Methods. Gene expression is denoted as fold difference relative to the WT GBS strain A909. Standard deviations areindicated.b �909 �rgfCAd/pRgfCA indicates a complementing plasmid encoding both RgfC and RgfA without the premature stop codon in RgfA.

FIG 5 Effect of increased expression of Sia-CPS and CspA in �rgfC mutant.(A) Quantity of sialic acid associated with the capsular polysaccharide of eachGBS strain was measured using HPLC. Data indicate that sialic acid levels arehigher in the �rgfCAd mutant (n 3; P 0.01 by Student’s t test). (B) Ten6-week-old male CD-1 mice were intravenously injected with 3 � 107 to 3 �108 CFU of the WT A909, �rgfCAd, or �rgfCAd �cspA strain. The Kaplan-Meier survival curve shows percent survival of mice after the infection. Notethat virulence of the �rgfCAd mutant is restored to WT levels in the �rgfCAd

�cspA double mutant (P 0.004 by log-rank test).

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this observation also is provided by the recent findings of Faralla etal. (21). The authors reported that the deletion of RgfC in the GBSserotype V strain CJB11, which encodes a functional RgfA, re-sulted in increased mortality of mice without significant differ-ences in bacterial dissemination (21). Notably, only 2 genesshowed altered transcription in the RgfC mutant compared to WTGBS CJB11 (see �rr17 in reference 21). Our studies using GBSCOH1 indicate that an RgfCA mutant does not exhibit alteredvirulence compared to the isogenic WT strains (see Fig. S3 in thesupplemental material). Collectively, these observations suggestthat RgfC regulation in GBS virulence is influenced by the pres-ence or absence of a functional RgfA and strain-specific genes.

Expression of the sialic acid capsular polysaccharide is criticalfor GBS virulence (46, 48). Our results suggest that RgfC regulatesproduction of the GBS capsule at both the transcriptional andposttranscriptional levels. Apart from changes in transcription ofgenes in the capsule operon, we identified aspartate phosphopep-tides corresponding to the tyrosine kinase CpsD (Table 3 and Fig.6). While tyrosine phosphorylation of CpsD at the C-terminalYGX motif has been shown previously to regulate capsule polymerlength in Streptococcus pneumoniae (51), the role of tyrosine phos-phorylation of CpsD in GBS capsule has not been established spe-cifically. As aspartate phosphorylation of CpsD was observed atthe YGD motif that also contains the putative tyrosine phosphor-ylation site (51), we speculate that aspartate phosphorylation im-pacts tyrosine phosphorylation and vice versa, as previously

shown with serine/threonine and aspartate phosphorylation forthe GBS response regulator CovR (13). Further studies on CpsDwill establish the role of tyrosine and aspartate phosphorylationon GBS capsule production and virulence.

Interestingly, we also identified an aspartate phosphopeptidecorresponding to a transcriptional regulator as a potential targetof RgfC (Table 3). Although this regulator is annotated as a hypo-thetical protein in the A909 genome, it shows homology to thexenobiotic response element family of transcriptional regulators.The potential for this protein to be an alternative DNA bindingresponse regulator of RgfC in the absence of RgfA is an excitinghypothesis that remains to be investigated.

The signal(s) sensed by RgfC remains unknown. The microar-ray data indicate that the deletion of RgfC results in a strong in-crease in transcripts of the phosphotransferase system (PTS) (seeTable S2 in the supplemental material), which aids in the uptake ofcarbohydrates, such as fructose, mannose, and sucrose, into thecell. Additionally, transcription levels are increased for uptakemachinery for both hydrophobic and polar amino acids. This sug-gests that RgfC responds to altered carbon metabolite levels, andconcurrent with this observation, we observed that the �rgfCAd

strain expressed greater levels of the carbon starvation proteinCtsA. In E. coli, this protein is thought to aid in the utilization ofpeptides during carbon starvation (52). Thus, another purpose ofthe increased expression of serine protease CspA is to increase theavailability of carbon in the form of peptides cleaved by this pro-

TABLE 3 Differential protein phosphorylation between WT GBS and the �rgfCAd mutanta

Phosphopeptide Protein Description

Aspartate phosphopeptides unique to RgfC expressing strains (A909 and/or the�rgfCAd/pRgfC complemented strain)

AdFVDILQNPSLMDQLPPLDMMK SAK_0723 XRE family transcriptional regulatorVSESVATYGDYGdYGNYGK SAK_1259 CpsD/tyrosine kinase

Aspartate phosphopeptides unique to �rgfCAd mutantVEdCSVLDIGTGSGAIAISLK SAK_1162 HemK family modification methylaseDLIFFVdER

S/T/Y/C phosphopeptides unique to WT A909 and/or the �rgfCAd/pRgfCcomplemented strain

GLMMGVAAMVTLLAPAVGPTYGGVIsGMLGWK SAK_1173 H� antiporter-2 (DHA2) family proteinQKQVDLCQNcyQIIK SAK_0590 ATP-dependent Clp protease, ATP-binding subunit ClpELVGAPPGYVGYEEAGQLtEKVR

S/T/Y phosphopeptides unique to �rgfCAd mutantQVILsEASR SAK_0420 Enoyl-(acyl-carrier-protein) reductase IIILYLQEIFAEEMsK SAK_1017 Hypothetical protein SAK_1017yFNLEDKLEsEGIHVR SAK_1756 TransketolaseFVLSAGHGsALLYSLLHLAGYDLSIDDLKKVSYVtIR SAK_0620 Hypothetical protein SAK_0620ELyIMIKQSGsIPEGTVIGDGK SAK_0435 PTS mannose/fructose/sorbose family transporter

subunit IIALLHGQVAtAWTPASKVKIsAQDEASNLMGK SAK_0586 Cell division protein DivIVAISAQDEAsNLMGKDLSGVTQTQIsLPFITAGSAGPLHLEMSLSR SAK_0147 Molecular chaperone DnaK

a Phosphopeptide enrichment was performed as described previously (50; also see Materials and Methods), and peptides were identified by mass spectrometric analyses. Thephosphorylation site was determined by phosphoRS. The phosphorylated aspartate, threonine, serine, tyrosine, or cysteine residue is indicated by d, t, s, y, or c, respectively. SAKnumbers correspond to the open reading frame of the gene in the GBS A909 genome (9). The proteins below were identified in two independent phosphopeptide enrichmentanalyses.

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tease. Interestingly, downregulation of the PTS recently was ob-served with GBS deficient in FspS/R (21), suggesting that TCShave differing roles in GBS metabolism.

It remains unclear if any evolutionary or selective advantage isconferred to GBS by the stop codon mutation in RgfA. As theincreased expression of virulence factors often can be associatedwith invasive disease, we speculate that decreased expression ofvirulence factors (e.g., due to the presence of RgfC in strains lack-ing a functional RgfA) promotes the existence of GBS in commen-sal niches, such as the rectovaginal tract of adult humans. Reportsof spontaneous mutations in TCS have been described in manybacteria that affect various phenotypic characteristics. For exam-ple, mutations in TCS have been shown to confer antibiotic resis-tance in certain strains of Streptococcus pneumoniae, Staphylococ-cus aureus, Pseudomonas aeruginosa, and Enterococcus faecium(53–56). Clinical isolates containing mutations in the TCS CovR/S(also known as CsrR/S) have been reported in GBS and group Astreptococci (GAS) (57, 58). As this TCS regulates the expressionof important virulence genes, mutation(s) in this locus signifi-cantly affects pathogenesis (11, 12, 59). Further studies are neededto elucidate the RgfC phosphosignaling cascade in GBS. Neverthe-less, our studies indicate that the acquisition of spontaneous mu-tations in signaling systems alters virulence gene expression innovel ways and provides insight into new mechanisms of generegulation that can occur in bacterial organisms.

ACKNOWLEDGMENTS

We are grateful to Francesco Bedogni and Rebecca Hodge (Seattle Chil-dren’s Research Institute) for their expert advice in harvesting the mousebrain. We thank James Connelly, Melissa de los Reyes, Mason Craig Bai-ley, and Nguyen-Thao BinhTran for technical support.

This work was supported by funding from the National Institutesof Health, grants R01AI100989, R56 AI070749, R01AI088255, andR21AI109222 to L.R., grant R01NS051247 from the NINDS/NIH to

K.S.D., and RO1GM088317 to W.A.T. Support for C.W. was provided byan NIH training grant (T32 AI07509; principal investigator, Lee AnnCampbell). We also thank the Center for Childhood Infections and Pre-maturity Research at Seattle Children’s Research Institute for strategicfunds.

The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes of Health.

REFERENCES1. Hoch JA, Silhavy TJ (ed). 1995. Two-component signal transduction.

American Society for Microbiology, Washington, DC.2. Beier D, Gross R. 2006. Regulation of bacterial virulence by two-

component systems. Curr Opin Microbiol 9:143–152. http://dx.doi.org/10.1016/j.mib.2006.01.005.

3. Calva E, Oropeza R. 2006. Two-component signal transduction systems,environmental signals, and virulence. Microb Ecol 51:166 –176. http://dx.doi.org/10.1007/s00248-005-0087-1.

4. Bassler B, Losick R. 2006. Bacterially speaking. Cell 125:237–246. http://dx.doi.org/10.1016/j.cell.2006.04.001.

5. Novick RP. 2003. Autoinduction and signal transduction in the regula-tion of staphylococcal virulence. Mol Microbiol 48:1429 –1449. http://dx.doi.org/10.1046/j.1365-2958.2003.03526.x.

6. Baker CJ, Edwards MW. 1995. Group B streptococcal infections, p 980 –1054. In Remington JS, Klein JO, Wilson CB, Baker CJ (ed), Infectiousdiseases of the fetus and newborn infant. W.B. Saunders, Philadelphia, PA.

7. Doran KS, Nizet V. 2004. Molecular pathogenesis of neonatal group Bstreptococcal infection: no longer in its infancy. Mol Microbiol 54:23–31.http://dx.doi.org/10.1111/j.1365-2958.2004.04266.x.

8. Rajagopal L. 2009. Understanding the regulation of group B streptococcalvirulence factors. Future Microbiol 4:201–221. http://dx.doi.org/10.2217/17460913.4.2.201.

9. Tettelin H, Masignani V, Cieslewicz MJ, Donati C, Medini D, WardNL, Angiuoli SV, Crabtree J, Jones AL, Durkin AS, Deboy RT,Davidsen TM, Mora M, Scarselli M, Margarit y Ros I, Peterson JD,Hauser CR, Sundaram JP, Nelson WC, Madupu R, Brinkac LM,Dodson RJ, Rosovitz MJ, Sullivan SA, Daugherty SC, Haft DH,Selengut J, Gwinn ML, Zhou L, Zafar N, Khouri H, Radune D,Dimitrov G, Watkins K, O’Connor KJ, Smith S, Utterback TR,White O, Rubens CE, Grandi G, Madoff LC, Kasper DL, Telford JL,Wessels MR, Rappuoli R, Fraser CM. 2005. Genome analysis of

FIG 6 Representative mass spectrum of an aspartate phosphopeptide unique to RgfC expressing GBS. The peptide, VSESVATYGDYGdYGNYGK from CspD,was identified uniquely in the RgfC-expressing strains (Table 3). The fragmentation of the precursor ion gives rise to peaks that can be mapped to the peptidesequence shown above the spectrum. In particular, both the y ion sequence and b ion sequence indicate the presence of a phosphorylated aspartate residue. Thisphosphorylated residue is highlighted by a lowercase “d” in the sequence.

Gendrin et al.

1086 iai.asm.org March 2015 Volume 83 Number 3Infection and Immunity

on May 3, 2020 by guest

http://iai.asm.org/

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nloaded from

multiple pathogenic isolates of Streptococcus agalactiae: implications forthe microbial “pan-genome.” Proc Natl Acad Sci U S A 102:13950 –13955.http://dx.doi.org/10.1073/pnas.0506758102.

10. Jiang SM, Cieslewicz MJ, Kasper DL, Wessels MR. 2005. Regulation ofvirulence by a two-component system in group B streptococcus. J Bacte-riol 187:1105–1113. http://dx.doi.org/10.1128/JB.187.3.1105-1113.2005.

11. Lamy MC, Zouine M, Fert J, Vergassola M, Couve E, Pellegrini E,Glaser P, Kunst F, Msadek T, Trieu-Cuot P, Poyart C. 2004. CovS/CovRof group B streptococcus: a two-component global regulatory system in-volved in virulence. Mol Microbiol 54:1250 –1268. http://dx.doi.org/10.1111/j.1365-2958.2004.04365.x.

12. Lembo A, Gurney MA, Burnside K, Banerjee A, de los Reyes M,Connelly JE, Lin WJ, Jewell KA, Vo A, Renken CW, Doran KS, Ra-jagopal L. 2010. Regulation of CovR expression in group B Streptococcusimpacts blood-brain barrier penetration. Mol Microbiol 77:431– 443.http://dx.doi.org/10.1111/j.1365-2958.2010.07215.x.

13. Lin WJ, Walthers D, Connelly JE, Burnside K, Jewell KA, Kenney LJ,Rajagopal L. 2009. Threonine phosphorylation prevents promoterDNA binding of the group B Streptococcus response regulator CovR.Mol Microbiol 71:1477–1495. http://dx.doi.org/10.1111/j.1365-2958.2009.06616.x.

14. Rajagopal L, Vo A, Silvestroni A, Rubens CE. 2006. Regulation ofcytotoxin expression by converging eukaryotic-type and two-componentsignalling mechanisms in Streptococcus agalactiae. Mol Microbiol 62:941–957. http://dx.doi.org/10.1111/j.1365-2958.2006.05431.x.

15. Patras KA, Wang NY, Fletcher EM, Cavaco CK, Jimenez A, Garg M,Fierer J, Sheen TR, Rajagopal L, Doran KS. 2013. Group B StreptococcusCovR regulation modulates host immune signalling pathways to promotevaginal colonization. Cell Microbiol 15:1154 –1167. http://dx.doi.org/10.1111/cmi.12105.

16. Poyart C, Pellegrini E, Marceau M, Baptista M, Jaubert F, Lamy MC,Trieu-Cuot P. 2003. Attenuated virulence of Streptococcus agalactiae de-ficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility todefensins and phagocytic cells. Mol Microbiol 49:1615–1625. http://dx.doi.org/10.1046/j.1365-2958.2003.03655.x.

17. Quach D, van Sorge NM, Kristian SA, Bryan JD, Shelver DW, DoranKS. 2009. The CiaR response regulator in group B Streptococcus pro-motes intracellular survival and resistance to innate immune defenses. JBacteriol 191:2023–2032. http://dx.doi.org/10.1128/JB.01216-08.

18. Rozhdestvenskaya AS, Totolian AA, Dmitriev AV. 2010. Inactivation ofDNA-binding response regulator Sak189 abrogates beta-antigen expres-sion and affects virulence of Streptococcus agalactiae. PLoS One 5:e10212.http://dx.doi.org/10.1371/journal.pone.0010212.

19. Al Safadi R, Mereghetti L, Salloum M, Lartigue MF, Virlogeux-PayantI, Quentin R, Rosenau A. 2011. Two-component system RgfA/C activatesthe fbsB gene encoding major fibrinogen-binding protein in highly viru-lent CC17 clone group B Streptococcus. PLoS One 6:e14658. http://dx.doi.org/10.1371/journal.pone.0014658.

20. Spellerberg B, Rozdzinski E, Martin S, Weber-Heynemann J, LuttickenR. 2002. rgf encodes a novel two-component signal transduction system ofStreptococcus agalactiae. Infect Immun 70:2434 –2440. http://dx.doi.org/10.1128/IAI.70.5.2434-2440.2002.

21. Faralla C, Metruccio MM, De Chiara M, Mu R, Patras KA, Muzzi A,Grandi G, Margarit I, Doran KS, Janulczyk R. 2014. Analysis of two-component systems in group B Streptococcus shows that RgfAC and thenovel FspSR modulate virulence and bacterial fitness. mBio 5:e00870 –14.http://dx.doi.org/10.1128/mBio.00870-14.

22. Madoff LC, Michel JL, Kasper DL. 1991. A monoclonal antibody iden-tifies a protective C-protein alpha-antigen epitope in group B strepto-cocci. Infect Immun 59:204 –210.

23. Martin TR, Rubens CE, Wilson CB. 1988. Lung antibacterial defensemechanisms in infant and adult rats: implications for the pathogenesis ofgroup B streptococcal infections in neonatal lung. J Infect Dis 157:91–100.http://dx.doi.org/10.1093/infdis/157.1.91.

24. Okada N, Geist RT, Caparon MG. 1993. Positive transcriptional controlof mry regulates virulence in the group A streptococcus. Mol Microbiol7:893–903. http://dx.doi.org/10.1111/j.1365-2958.1993.tb01180.x.

25. Horton RM. 1995. PCR-mediated recombination and mutagenesis. SOE-ing together tailor-made genes. Mol Biotechnol 3:93–99.

26. Chaffin DO, Beres SB, Yim HH, Rubens CE. 2000. The serotype of typeIa and III group B streptococci is determined by the polymerase genewithin the polycistronic capsule operon. J Bacteriol 182:4466 – 4477. http://dx.doi.org/10.1128/JB.182.16.4466-4477.2000.

27. Rajagopal L, Clancy A, Rubens CE. 2003. A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversiblyphosphorylate an inorganic pyrophosphatase and affect growth, cell seg-regation, and virulence. J Biol Chem 278:14429 –14441. http://dx.doi.org/10.1074/jbc.M212747200.

28. Husmann LK, Scott JR, Lindahl G, Stenberg L. 1995. Expression of theArp protein, a member of the M protein family, is not sufficient to inhibitphagocytosis of Streptococcus pyogenes. Infect Immun 63:345–348.

29. Chaffin DO, Rubens CE. 1998. Blue/white screening of recombinantplasmids in Gram-positive bacteria by interruption of alkaline phospha-tase gene (phoZ) expression. Gene 219:91–99. http://dx.doi.org/10.1016/S0378-1119(98)00396-5.

30. National Research Council. 2011. Guide for the care and use of laboratoryanimals, 8th ed. National Academies Press, Washington, DC.

31. Jones AL, Knoll KM, Rubens CE. 2000. Identification of Streptococcusagalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis. Mol Microbiol 37:1444 –1455. http://dx.doi.org/10.1046/j.1365-2958.2000.02099.x.

32. Doran KS, Liu GY, Nizet V. 2003. Group B streptococcal beta-hemolysin/cytolysin activates neutrophil signaling pathways in brain en-dothelium and contributes to development of meningitis. J Clin Investig112:736 –744. http://dx.doi.org/10.1172/JCI17335.

33. Doran KS, Engelson EJ, Khosravi A, Maisey HC, Fedtke I, Equils O,Michelsen KS, Arditi M, Peschel A, Nizet V. 2005. Blood-brain barrierinvasion by group B Streptococcus depends upon proper cell-surface an-choring of lipoteichoic acid. J Clin Investig 115:2499 –2507. http://dx.doi.org/10.1172/JCI23829.

34. Stins MF, Prasadarao NV, Zhou J, Arditi M, Kim KS. 1997. Bovine brainmicrovascular endothelial cells transfected with SV40-large T antigen: de-velopment of an immortalized cell line to study pathophysiology of CNSdisease. In Vitro Cell Dev Biol Anim 33:243–247. http://dx.doi.org/10.1007/s11626-997-0042-1.

35. Burnside K, Lembo A, Harrell MI, Gurney M, Xue L, Binhtran NT,Connelly JE, Jewell KA, Schmidt BZ, de Los Reyes M, Tao WA, DoranKS, Rajagopal L. 2011. Serine/threonine phosphatase Stp1 mediates post-transcriptional regulation of hemolysin, autolysis, and virulence of groupB Streptococcus. J Biol Chem 286:44197– 44210. http://dx.doi.org/10.1074/jbc.M111.313486.

36. Silvestroni A, Jewell KA, Lin WJ, Connelly JE, Ivancic MM, Tao WA,Rajagopal L. 2009. Identification of serine/threonine kinase substrates inthe human pathogen group B streptococcus. J Proteome Res 8:2563–2574.http://dx.doi.org/10.1021/pr900069n.

37. Iliuk AB, Martin VA, Alicie BM, Geahlen RL, Tao WA. 2010. In-depthanalyses of kinase-dependent tyrosine phosphoproteomes based on metalion-functionalized soluble nanopolymers. Mol Cell Proteomics 9:2162–2172. http://dx.doi.org/10.1074/mcp.M110.000091.

38. Iliuk A, Tao WA. 2009. Quantitative phospho-proteomics based on sol-uble nanopolymers. Methods Mol Biol 527:117–129, ix. http://dx.doi.org/10.1007/978-1-60327-834-8_10.

39. Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, MannM. 2006. Global, in vivo, and site-specific phosphorylation dynamics insignaling networks. Cell 127:635– 648. http://dx.doi.org/10.1016/j.cell.2006.09.026.

40. Taus T, Kocher T, Pichler P, Paschke C, Schmidt A, Henrich C,Mechtler K. 2011. Universal and confident phosphorylation site localiza-tion using phosphoRS. J Proteome Res 10:5354 –5362. http://dx.doi.org/10.1021/pr200611n.

41. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: apractical and powerful approach to multiple testing. J R Stat Soc Ser B57:289 –300.

42. Jones AL, Needham RH, Clancy A, Knoll KM, Rubens CE. 2003.Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanismfor evasion of immune clearance. Mol Microbiol 47:247–256. http://dx.doi.org/10.1046/j.1365-2958.2003.03297.x.

43. Lancefield RC, McCarty M, Everly WN. 1975. Multiple mouse-protective antibodies directed against group B streptococci. Special refer-ence to antibodies effective against protein antigens. J Exp Med 142:165–179.

44. Harris TO, Shelver DW, Bohnsack JF, Rubens CE. 2003. A novel strep-tococcal surface protease promotes virulence, resistance to opsonophago-cytosis, and cleavage of human fibrinogen. J Clin Investig 111:61–70. http://dx.doi.org/10.1172/JCI16270.

45. Bryan JD, Shelver DW. 2008. Streptococcus agalactiae CspA is a serine

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protease that inactivates chemokines. J Bacteriol 191:1847–1854. http://dx.doi.org/10.1128/JB.01124-08.

46. Wessels MR, Rubens CE, Benedi VJ, Kasper DL. 1989. Definition of abacterial virulence factor: sialylation of the group B streptococcal capsule.Proc Natl Acad Sci U S A 86:8983– 8987. http://dx.doi.org/10.1073/pnas.86.22.8983.

47. Rubens CE, Wessels MR, Heggen LM, Kasper DL. 1987. Transposonmutagenesis of type III group B streptococcus: correlation of capsule ex-pression with virulence. Proc Natl Acad Sci U S A 84:7208 –7212. http://dx.doi.org/10.1073/pnas.84.20.7208.

48. Marques MB, Kasper DL, Pangburn MK, Wessels MR. 1992. Preventionof C3 deposition by capsular polysaccharide is a virulence mechanism oftype III group B streptococci. Infect Immun 60:3986 –3993.

49. Chaffin DO, Mentele LM, Rubens CE. 2005. Sialylation of group Bstreptococcal capsular polysaccharide is mediated by cpsK and is requiredfor optimal capsule polymerization and expression. J Bacteriol 187:4615–4626. http://dx.doi.org/10.1128/JB.187.13.4615-4626.2005.

50. Burnside K, Lembo A, de Los Reyes M, Iliuk A, Binhtran NT, ConnellyJE, Lin WJ, Schmidt BZ, Richardson AR, Fang FC, Tao WA, RajagopalL. 2010. Regulation of hemolysin expression and virulence of Staphylo-coccus aureus by a serine/threonine kinase and phosphatase. PLoS One5:e11071. http://dx.doi.org/10.1371/journal.pone.0011071.

51. Bender MH, Cartee RT, Yother J. 2003. Positive correlation betweentyrosine phosphorylation of CpsD and capsular polysaccharide produc-tion in Streptococcus pneumoniae. J Bacteriol 185:6057– 6066. http://dx.doi.org/10.1128/JB.185.20.6057-6066.2003.

52. Schultz JE, Matin A. 1991. Molecular and functional characterization ofa carbon starvation gene of Escherichia coli. J Mol Biol 218:129 –140. http://dx.doi.org/10.1016/0022-2836(91)90879-B.

53. Moscoso M, Domenech M, Garcia E. 28 June 2010. Vancomycin toler-

ance in clinical and laboratory Streptococcus pneumoniae isolates dependson reduced enzyme activity of the major LytA autolysin or cooperationbetween CiaH histidine kinase and capsular polysaccharide. Mol Micro-biol http://dx.doi.org/10.1111/j.1365-2958.2010.07271.x.

54. Katayama Y, Murakami-Kuroda H, Cui L, Hiramatsu K. 2009. Selectionof heterogeneous vancomycin-intermediate Staphylococcus aureus by imi-penem. Antimicrob Agents Chemother 53:3190 –3196. http://dx.doi.org/10.1128/AAC.00834-08.

55. Lau CH, Fraud S, Jones M, Peterson SN, Poole K. 2013. Mutationalactivation of the AmgRS two-component system in aminoglycoside-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:2243–2251. http://dx.doi.org/10.1128/AAC.00170-13.

56. Depardieu F, Courvalin P, Msadek T. 2003. A six amino acid deletion,partially overlapping the VanSB G2 ATP-binding motif, leads to con-stitutive glycopeptide resistance in VanB-type Enterococcus faecium.Mol Microbiol 50:1069–1083. http://dx.doi.org/10.1046/j.1365-2958.2003.03771.x.

57. Sendi P, Johansson L, Dahesh S, Van-Sorge NM, Darenberg J, NorgrenM, Sjolin J, Nizet V, Norrby-Teglund A. 2009. Bacterial phenotypevariants in group B streptococcal toxic shock syndrome. Emerg Infect Dis15:223–232. http://dx.doi.org/10.3201/eid1502.080990.

58. Miller AA, Engleberg NC, DiRita VJ. 2001. Repression of virulence genesby phosphorylation-dependent oligomerization of CsrR at target promot-ers in S. pyogenes. Mol Microbiol 40:976 –990. http://dx.doi.org/10.1046/j.1365-2958.2001.02441.x.

59. Heath A, DiRita VJ, Barg NL, Engleberg NC. 1999. A two-componentregulatory system, CsrR-CsrS, represses expression of three Streptococcuspyogenes virulence factors, hyaluronic acid capsule, streptolysin S, andpyrogenic exotoxin B. Infect Immun 67:5298 –5305.

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