The Streptococcus agalactiae Stringent Response EnhancesVirulence and Persistence in Human Blood

Thomas A. Hooven,a Andrew J. Catomeris,b* Maryam Bonakdar,b Luke J. Tallon,c Ivette Santana-Cruz,c Sandra Ott,c

Sean C. Daugherty,c* Hervé Tettelin,c Adam J. Ratnerb,d

aDepartment of Pediatrics, Columbia University, New York, New York, USAbDepartment of Pediatrics, New York University, New York, New York, USAcInstitute for Genome Sciences, University of Maryland, Baltimore, Maryland, USAdDepartment of Microbiology, New York University, New York, New York, USA

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious in-fections in neonates. We previously reported a transposon sequencing (Tn-seq) sys-tem for performing genomewide assessment of gene fitness in GBS. In order toidentify molecular mechanisms required for GBS to transition from a mucosal com-mensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909with human whole blood. Our analysis identified 16 genes conditionally essential forGBS survival in blood, of which 75% were members of the capsular polysaccharide(cps) operon. Among the non-cps genes identified as conditionally essential was relA,which encodes an enzyme whose activity is central to the bacterial stringent re-sponse—a conserved adaptation to environmental stress. We used blood coincuba-tion studies of targeted knockout strains to confirm the expected growth defects ofGBS deficient in capsule or stringent response activation. Unexpectedly, we foundthat the relA knockout strains demonstrated decreased expression of �-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Further-more, chemical activation of the stringent response with serine hydroxamate in-creased �-hemolysin/cytolysin expression. To establish a mechanism by which thestringent response leads to increased cytotoxicity, we performed transcriptome se-quencing (RNA-seq) on two GBS strains grown under stringent response or controlconditions. This revealed a conserved decrease in the expression of genes in the ar-ginine deiminase pathway during stringent response activation. Through coincuba-tion with supplemental arginine and the arginine antagonist canavanine, we showthat arginine availability is a determinant of GBS cytotoxicity and that the pathwaybetween stringent response activation and increased virulence is arginine depen-dent.

KEYWORDS Streptococcus agalactiae, bloodstream infections, Tn-seq

Streptococcus agalactiae (group B Streptococcus [GBS]) is a common adult intestinaland vaginal commensal that also causes neonatal sepsis, pneumonia, and menin-

gitis (1, 2). It is the leading cause of infectious neonatal mortality in the United States(3). Enhanced understanding of the host-pathogen interactions that permit GBS toconvert from a commensal to an invasive lifestyle would help advance the develop-ment of improved preventative and therapeutic approaches.

GBS expresses numerous virulence factors, although there is variability in expressionamong individual strains (4–9). One well-studied virulence factor is the pigmentedornithine-rhamnopolyene �-hemolysin/cytolysin (�HC; also referred to as granadaene)(10). Although its exact molecular mechanism is not understood, �HC has been shownto be cytotoxic to a variety of human cells and to contribute to virulence in several

Received 28 August 2017 Returned formodification 6 October 2017 Accepted 30October 2017

Accepted manuscript posted online 6November 2017

Citation Hooven TA, Catomeris AJ, BonakdarM, Tallon LJ, Santana-Cruz I, Ott S, DaughertySC, Tettelin H, Ratner AJ. 2018. TheStreptococcus agalactiae stringent responseenhances virulence and persistence in humanblood. Infect Immun 86:e00612-17. https://doi.org/10.1128/IAI.00612-17.

Editor Nancy E. Freitag, University of Illinois atChicago

Copyright © 2017 Hooven et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.

Address correspondence to Adam J. Ratner,Adam.Ratner@nyumc.org.

* Present address: Andrew J. Catomeris,Georgetown University School of Medicine,Washington, DC, USA; Sean C. Daugherty,Ultragenyx Pharmaceutical, Novato,California, USA.

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animal models of disease (11–15). There is considerable variability in �HC expressionamong GBS strains, even among pathogenic strains (16). Within individual strains,changes in the environment—such as shifts in pH or temperature—affect �HC expres-sion (17, 18).

We recently reported the development and validation of a transposon sequencing(Tn-seq) method for performing unbiased, whole-genome identification of essential orconditionally essential (CE) GBS genes (19). Tn-seq uses next-generation sequencing(NGS) of a saturated transposon mutant library to compare transposon insertions inlibrary bacteria grown under experimental conditions to control library outgrowth.Genes with decreased transposon insertion densities after the experimental exposureare likely essential for bacterial growth under that condition; the decrease in trans-poson insertions detected indicates that mutants bearing knockouts of those genes,which were present in the starting library, have died off (20, 21).

In this study, we apply our Tn-seq method to identifying gene products necessaryfor GBS survival in human whole blood. We show that the GBS polysaccharide capsuleis CE for survival in blood, as is RelA, a ribosome-associated GTP pyrophosphokinase.RelA is a central effector of the bacterial stringent response (SR), a conserved, globaltranscriptional adaptation to environmental stress (22–24). Using transcriptomics andconfirmatory coincubation studies, we show that in addition to promoting GBS persis-tence in human blood, activation of the stringent response enhances �HC expressionthrough an arginine-mediated pathway and transcription of genes involved in argininemetabolism is implicated in �HC expression variability among different GBS strains.

RESULTSGBS Tn-seq in whole blood identifies capsule and RelA as conditionally essen-

tial. In order to maximize the resolution of our Tn-seq method, we combined threeTn-seq-compatible GBS transposon mutant libraries to generate a pooled master libraryin a background of the pathogenic GBS serotype Ia strain A909. The generation of thelibraries and our basic Tn-seq methodology have been described previously (19).

We performed library outgrowth for 6 h in five samples of fresh whole blood fromthree healthy adult volunteers and one control condition of selective medium. Then,bacterial genomic DNA was purified from each sample, digested with MmeI, ligated tobarcoded adapters, and used as the template for PCR. The resultant amplicons werepurified and sequenced by NGS. The reads were trimmed of all transposon and adaptersequences, leaving only 16-nucleotide (nt) GBS genomic-DNA sequences, which werealigned to the A909 genome. These alignments were then analyzed using ESSENTIALS,an open-access Tn-seq bioinformatics tool that compares experimental and controlalignments, in order to identify CE genes from the experimental condition (25).

Genomewide results from our Tn-seq analysis are presented as a Circos plot in Fig.1 (26). After passage through blood or, in the case of the control sample, tryptic soybroth (TSB) with erythromycin (Erm) (TSB Erm) selection, sequencing of DNA from oursix samples identified 62,217 unique flanking transposon insertion sites.

The number of transposon insertions detected within each gene was highly repro-ducible among our five blood coincubation replicates (Fig. 1, rings 1 to 5). Comparisonbetween TA site insertions detected in the control, under the broth-only coincubationcondition, and in the five blood coincubation samples (Fig. 1, rings 7 to 8) identifiedseveral regions of statistically significant divergence, where fewer TA site insertionswere detected in the blood coincubation samples than in the control sample, indicatingthat these regions were CE for GBS survival in human whole blood (Fig. 1, ring 9).

ESSENTIALS generates a plot of kernel function density versus log2-fold change (LFC;actual versus expected transposon insertions) for the genome (25). This plot providesa visualization of the relative number of genes that have fewer than expected trans-poson insertions in the experimental data set. The LFC value at the local minimumbetween the rightmost peak (which represents genes that have approximately theexpected number of insertions) and the leftmost peak (representing genes with fewerthan expected transposon insertions) can be used as a cutoff between genes that are

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putatively dispensable and those that are CE under the experimental growth condition.In our experiment, the leftmost peak—representing CE genes for blood coincubation—was short, indicating that among genes that are nonessential for growth in medium,only a small set are CE for GBS survival in blood (Fig. 2A). Figure 1, ring 9, shows theESSENTIALS LFC values for all genes in the A909 genome. Genomewide Tn-seq data arepresented in Data Set S1 in the supplemental material.

Table 1 lists the 16 GBS genes with LFC values below the cutoff of �4.1 identifiedby ESSENTIALS as the threshold between dispensable and CE genes. Of these, 75% arepart of the capsular polysaccharide synthesis (cps) operon (27). The relative paucity oftransposon insertions in the cps operon is visible in Fig. 1, ring 9. The gene with thelowest LFC score was cpsA, which encodes an important regulator of capsule synthesisand whose proper function is known to be important in promoting GBS survival inhuman blood (28). Of the four remaining genes with subthreshold LFC values,SAK_0483 encodes an R3H domain-containing protein that is predicted to interact withsingle-stranded DNA or RNA but has not been studied experimentally (29). SAK_1895encodes a predicted carbohydrate transporter subunit. SAK_0186, which encodes theIgA-binding � antigen, is found in serotype Ia, Ib, II, and some serotype III GBS strains(30, 31). Its upregulation in response to exposure to human blood, serum, and condi-

FIG 1 Circos plot of GBS whole blood Tn-seq results. Rings 1 to 5 show detected transposon insertioncounts per gene (white, lower; red, higher) for the five blood coincubation samples analyzed. Ring 6 showsbaseline fitness previously determined for each gene (green, nonessential; red, essential; yellow, critical;gray, undetermined) (19). Rings 7 and 8 show log-transformed unique transposon insertion counts at eachTA dinucleotide site for the control coincubation sample (ring 7, black) and the mean of all five experi-mental samples (ring 8, red). Ring 9 illustrates LFC for each gene, heat mapped for the adjusted P valueassigned by ESSENTIALS (red, lower; green, higher). The five loci that had LFC values below the CE thresholdare labeled.

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tions associated with fetal infection has been described, as has its putative role invirulence (32–35).

Coincubation studies validate the Tn-seq prediction that capsule and RelAare conditionally essential for GBS survival in blood. SAK_1900 encodes the GTPpyrophosphokinase RelA, which is a central mediator of the bacterial SR, a conservedtranscriptional adaptation to environmental stress (24). RelA is a ribosome-associatedenzyme that detects stalled protein translation, which can be the result of depletedmicronutrients, antibiotic exposure, or immunologic pressure, such as exposure toantimicrobial peptides (23, 36, 37). In response to stalled translation, RelA phosphory-lates GTP to generate the alarmone molecules guanosine tetra- and pentaphosphate[(p)ppGpp], which have been shown to act on multiple intracellular targets and secondmessengers, triggering global transcriptional changes, metabolic adjustment, and insome bacterial species, increased virulence (38–44).

Since studies of the GBS SR have not been reported and since it is a known mediatorof virulence in other pathogenic bacteria, we decided to pursue further investigation ofthis pathway.

To validate the predictions made by our Tn-seq experiments, we focused on theroles of the cps operon and RelA. The relative transposon insertion densities in CE cps

FIG 2 Conditionally essential genes for GBS whole blood survival. (A) Density versus LFC plot generated by ESSENTIALS, with the LFC threshold betweennonessential and CE genes (�4.1) indicated. (B) Mean unique transposon insertions within CE genes of the cps operon (gray bars) and relA (white bar) detectedafter blood coincubation relative to those in control outgrowth culture, normalized to the genomewide ratio of experimental to control transposon insertiondetections (black bar). ****, P � 0.001, exact test based on the negative binomial distribution model in EdgeR incorporated in the ESSENTIALS package. (C)Fractional survival of A909 WT, ΔcpsE, ΔrelA, and ΔrelA�pDC123:relA after coincubation with heparinized whole blood. Each coincubation was repeated at leastthree times. *, P � 0.05, t test with Bonferroni correction for multiple comparisons. Data represent mean values of all replicates, with error bars indicatingstandard errors of the means.

TABLE 1 GBS gene products conditionally essential for survival in human blood andcorresponding ESSENTIALS Log2 FC values

Gene locus Gene producta Log2 FC

SAK_1262 Regulatory protein CpsA �7.34SAK_1255 Capsular polysaccharide synthesis protein CpsH �6.24SAK_1251 Polysaccharide biosynthesis protein CpsL �5.65SAK_0483 R3H domain-containing protein �5.64SAK_1254 Capsular polysaccharide biosynthesis protein �5.42SAK_1259 Tyrosine-protein kinase CpsD �5.23SAK_1260 Capsular polysaccharide biosynthesis protein CpsC �5.15SAK_1249 UDP-N-acetylglucosamine-2-epimerase NeuC �5.03SAK_1900 GTP pyrophosphokinase RelA �4.97SAK_1895 PTS system transporter subunit IIA �4.92SAK_1258 Glycosyl transferase CpsE �4.83SAK_1253 Capsular polysaccharide biosynthesis protein CpsJ �4.76SAK_1248 NeuD protein �4.70SAK_0186 IgA-binding � antigen �4.38SAK_1256 Polysaccharide biosynthesis protein CpsG �4.36SAK_1257 Polysaccharide biosynthesis protein CpsF �4.32aThe products of genes in the capsular polysaccharide locus are in boldface.

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operon genes and relA are shown in Fig. 2B; all were significantly below the genome-wide insertion density. We generated ΔcpsE and ΔrelA knockout (KO) strains in an A909background and complemented the ΔrelA strain with the full-length relA codingsequence in trans (ΔrelA�pDC123:relA). In whole blood coincubation studies with theΔcpsE and ΔrelA strains, we observed the expected survival impairment, which waspartially rescued by complementation in the case of the ΔrelA strain (Fig. 2C).

SR activation increases GBS �HC expression. We observed that ΔrelA colonieswere less pigmented than wild-type (WT) colonies, suggesting decreased �HC expres-sion. The effect was especially pronounced when the KO strains were grown inpigment-enhancing new Granada medium (45). To confirm that this observation wasneither strain-specific nor an epiphenomenon unrelated to the SR, we generated relAKO strains in the pathogenic, hyperhemolytic serotype V strain 10/84 (46), and we alsogenerated A909 and 10/84 KO strains lacking codY (ΔcodY strains), which encodes aglobal transcription factor whose activity is regulated by the balance of GTP and(p)ppGpp and which is a crucial component of the SR (39, 40, 47–50). These additionalKO strains all had decreased pigmentation relative to that of the WT in new Granadamedium, reflecting decreased �HC expression (Fig. 3A).

We performed hemolysis assays that confirmed functionally that A909 ΔrelA haddecreased cytotoxicity compared to those of the WT and ΔrelA�pDC123:relA strains(Fig. 3B). Next, we performed coincubation studies with GBS and the SR inducer serinehydroxamate (SHX). Dose-dependent �HC expression was easily visualized in 10/84

FIG 3 The stringent response modulates GBS �HC expression and hemolytic activity. (A) A909 and 10/84WT and SR mutants were grown overnight in new Granada medium with appropriate antibiotic selection.The cultures were normalized for OD600 and volume, pelleted, resuspended in 100 �l PBS, and photo-graphed in a 96-well plate. (B) A909 ΔrelA shows decreased hemolysis relative to the results for the WTand ΔrelA�pDC123:relA strains. (C) WT A909 grown under SR-activating conditions with supplementalSHX shows enhanced hemolysis over bacteria grown in TSB or A909 ΔcylE, which does not produce �HC.All hemolysis experiments were performed in triplicate and repeated at least twice. The percentage ofhemolysis is relative to the result for a 1% Triton X-100 positive-control solution. Histograms show meanvalues, with error bars illustrating standard errors of the means. ***, P � 0.005, and ****, P � 0.0001, t testwith Bonferroni correction for multiple comparisons.

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coincubated with SHX, indicating that chemical SR induction leads to increased �HCproduction in this strain (Fig. S1). While hyperpigmentation was not observed in A909,a hemolysis assay after SHX coincubation demonstrated increased cytotoxicity (Fig. 3C).The same SHX coincubation was performed with an A909 ΔcylE KO strain deficient in�HC expression, which did not lead to increased hemolysis, indicating that the in-creased �HC expression observed under SR conditions is the cause of enhanced toxicity(Fig. 3C). Coincubation of WT A909 with equimolar concentrations of L-serine, which ischemically similar to SHX, did not enhance hemolysis, nor did SHX in the absence ofGBS (Fig. 3C).

To ensure that the SR toxicity effect was not specific to erythrocytes, we studiedcytotoxicity against HeLa cells by measuring lactate dehydrogenase (LDH) release aftercoincubation with A909 ΔrelA and ΔcodY strains and observed significantly decreasedtoxicity from both KO strains (Fig. S2).

We performed thin-layer chromatography with A909 ΔrelA, ΔcodY, and WT strainsgrown under SR and non-SR conditions to confirm that GBS produces ppGpp, that itslevels increase in response to SHX, and that our ΔrelA strain produces less ppGpp thanthe WT. We observed decreased but detectable ppGpp levels in both KO strains grownunder SR conditions relative to the level in the WT (Fig. 4). This suggests that in GBS,as in other Firmicutes, accessory (p)ppGpp synthases are active, preventing the com-plete absence of ppGpp in A909 ΔrelA (51). It also suggests possible direct reciprocalinteractions between CodY and RelA or one of the accessory synthases, which wouldexplain the decreased ppGpp levels in A909 ΔcodY.

RNA-seq reveals decreased arginine deiminase pathway activity after SR acti-vation. To explore how SR activation leads to increased �HC expression, we performedwhole-genome RNA-seq on RNA isolated from GBS grown in the presence of SHX or inTSB. We used A909 and the hyperhemolytic strain 10/84. This approach also allowed usto compare baseline gene expression differences between the two strains undernon-SR conditions.

Summary data for the RNA-seq run is presented in Data Set S2. Overall, there wasexcellent coverage of the sequencing reads from both strains under SR and controlgrowth conditions, with mapping to 100% of coding sequences in all replicates andreads per kilobase per million mapped reads (RPKM) scores between 667 and 755.

FIG 4 (A) Thin-layer chromatography ppGpp detection from GBS. (B) Densitometry of the ppGpp spotsfrom the autoradiograph presented was performed with ImageJ software. The percentage of maximalppGpp is relative to the result for the A909 WT SHX-positive condition.

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A909 and 10/84 showed significant between- and within-strain variation in overallgene expression under control and SR conditions, with approximately balanced up- anddownregulation of genes induced by SHX (Fig. 5A). We performed gene set enrichmentanalysis of genes whose transcription was significantly up- or downregulated by SRgrowth (52). This demonstrated that arginine deiminase pathway genes were signifi-cantly overrepresented among the set of genes downregulated by SR in both strains.Furthermore, when we compared whole-genome expression between 10/84 and A909grown under non-SR conditions, we identified arginine deiminase pathway genes assignificantly more highly expressed in A909 than in 10/84 (Table 2).

Combining the results of these analyses suggested that SR may trigger downregu-lation of genes in the arginine deiminase pathway, which converts arginine to ornithineand carbamoyl-phosphate (Fig. 5B). The fact that we observed the same pattern ofrelative arginine deiminase pathway downregulation in 10/84, which overproduces�HC compared to its production by A909, suggested that changes in arginine deimi-nase pathway activity, as well as the resultant changes in intracellular arginine levels,might represent a common mechanism of GBS �HC regulation.

Arginine availability modulates GBS �HC expression. We performed coincuba-tion studies to test the hypothesis that arginine availability is a regulator of GBS �HCexpression. The effect of supplemental arginine on GBS �HC was grossly visible, with

FIG 5 RNA-seq reveals conserved downregulation of the arginine deiminase pathway during SR activation and in comparison between 10/84 and A909 atbaseline. (A) Heat map with hierarchical clustering illustrating gene expression changes detected by RNA-seq in A909 and 10/84 during SR activation relativeto control growth. All genes that showed �2-fold expression changes (up- or downregulation) in either strain are included. (B) Illustration of the argininedeiminase pathway, with SR versus non-SR and 10/84 versus A909 baseline expression in TSB indicated by the heat-mapped rectangles above each gene inthe pathway. The complete list of genes with �2-fold expression changes as a result of SR activation is in Data Set S5. Normalized read counts for cross-straincomparison of A909 and 10/84 under SR and non-SR growth conditions are in Data Set S6.

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increased pigmentation under the coincubation condition relative to that in thecontrol; the effect was seen for strains A909 and 10/84 (Fig. 6A).

To test for a functional effect of arginine availability, we coincubated A909 witharginine and the competitive arginine inhibitor canavanine and then performed he-molysis assays with the resultant GBS samples (53, 54). We observed increased hemo-lysis by GBS grown in the presence of arginine and suppression when canavanine wasincluded in the coincubation. Neither arginine nor canavanine alone had any hemolyticeffect (Fig. 6B).

TABLE 2 Gene set enrichment analysis for KEGG classes significantly differently expressed at baseline or following stringent responseactivation in 10/84 and A909

Strain(s) and condition, KEGGclass downregulated No. of hits Class size P value

AdjustedP value Description

10/84 vs A909 at baseline330 6 12 6.10E�06 0.00011 Arginine and proline metabolism: arcA, argF, arc, argG,

SAK_2064 (putative duplicated arcC), SAK_2065(putative duplicated argF)

500 6 35 5.20E�03 0.03334 Starch and sucrose metabolism790 3 8 5.60E�03 0.03334 Folate biosynthesis920 2 3 7.40E�03 0.03334 Sulfur metabolism190 2 4 1.40E�02 0.04309 Oxidative phosphorylation

A909 under SRa

330 2 12 0.021 0.037 Arginine and proline metabolism: arcA, argF564 2 6 0.005 0.024 Glycerophospholipid metabolism640 2 10 0.015 0.034 Propanoate metabolism650 2 7 0.007 0.024 Butanoate metabolism

1084 under SR220 2 9 0.0017 0.0017 Arginine biosynthesis: arcA, glnA240 6 42 �1E�12 �1E�12 Pyrimidine metabolism250 4 13 �1E�12 �1E�12 Alanine, aspartate, and glutamate metabolism

aSR, stringent response.

FIG 6 Arginine availability modulates �HC expression and the hemolytic response induced by the SR. (A)A909 and 10/84 WT were grown overnight in TSB with 50 mM arginine or vehicle control. The cultureswere normalized for OD600 and volume, pelleted, resuspended in 100 �l PBS, and photographed in a96-well plate. (B) A909 grown overnight with supplemental arginine (10 mM) demonstrates increasedhemolytic activity, which is reversed by canavanine (1 mM). Canavanine also reverses the increasedcytotoxicity induced by SR induction with SHX. The hemolysis experiment was performed in triplicateand repeated twice. The percentage of hemolysis is relative to the result for a 1% Triton X-100positive-control solution. Histogram bars show mean values, with error bars illustrating standard errorsof the means. ***, P � 0.005, and ****, P � 0.0001, t test with Bonferroni correction for multiplecomparisons.

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To investigate whether the increased hemolysis observed after induction of the SRdepends on arginine availability, we repeated the coincubation with SHX, this timeadding an SHX-plus-canavanine condition. We found that 1 mg/ml SHX and 1 mMcanavanine was lethal to GBS (data not shown), and so for this experiment, we loweredthe SHX concentration to 0.5 mg/ml, which still triggered increased hemolysis whileallowing GBS to grow to normal density when canavanine was added. Functionallylimiting arginine availability with canavanine reduced the enhanced hemolysis ob-served with SHX coincubation, suggesting that the mechanism by which the SR leadsto increased �HC depends on arginine availability (Fig. 6B).

DISCUSSION

GBS is a common human commensal organism, with persistent rectovaginal colo-nization occurring in approximately 25% of asymptomatic adults (55). In order to causeneonatal sepsis, GBS must traverse distinct microenvironments, potentially includingcervical mucus, amniotic fluid, the respiratory mucosa, and blood (56). Pathogenesismay also require GBS to survive transcellular host cell passage (7). We have shown thatthe SR may contribute to GBS pathogenicity in two ways: by enhancing resistance tokilling in the bloodstream and by increasing the expression of the pigmented cytotoxin�HC. To our knowledge, this is the first published investigation of the GBS SR.

Our Tn-seq analysis of GBS grown in blood also revealed an important role for theGBS polysaccharide capsule in promoting survival. In addition to serving as a physicalbarrier against immune factors, the capsule anchors several known surface-associatedimmune inactivation proteins, such as C5a peptidase and the IgA-binding � antigen,which also emerged from our screen as CE for blood survival (31, 32, 57, 58). Given thisbackground, identification of the cps locus as CE for blood survival is not surprising, butsupports the validity of our Tn-seq system. Two other genes identified as CE—the R3Hdomain-containing protein encoded by SAK_0483 and the carbohydrate transportersubunit encoded by SAK_1895—are not characterized and warrant future study.

The SR can be activated by a variety of environmental stresses, many of which arelikely to be encountered in the blood. Nutrient deprivation, exposure to antimicrobialpeptides or antibiotics, and phagosome exposure can all activate the SR in Firmicutesrelated to GBS (23, 44, 59). RelA is likely not the sole enzyme involved in (p)ppGpphomeostasis in GBS. Several accessory RelA-related proteins have been described inclosely related Streptococcus species (60, 61), and GBS has homologous genes (data notshown). While we have not performed a comprehensive assessment of the roles ofthose accessory enzymes for this report, further investigation may be worthwhile infuture studies.

Widespread transcriptional changes occur in bacteria when the SR is activated, andthe changes that promote GBS survival in blood are likely multifactorial and not limitedto the arginine deiminase pathway genes that contribute to increased �HC expression.However, the two effects of SR activation that we report—prolonged survival in bloodand increased cytotoxin expression— can both be viewed as adaptive responses to afundamentally inhospitable environment. Based on our findings, the SR allows GBS tosurvive longer in blood, while also upregulating a cytotoxin that has been shown topromote invasion across anatomical barriers, potentially allowing the bacteria to escapethe bloodstream into a less immunologically active space (5, 14, 62). We did not pursuea firm explanation for why complementation of the relA KO strain only provided partialrescue of the WT phenotype, but existing studies suggest that the wild-type stringentresponse is controlled by a finely tuned and interdependent network of (p)ppGppsynthases and hydrolases, as well as second messengers like CodY (40). We speculatethat if the transcription of the relA gene off the complementation vector is eitherslightly less or slightly more than its transcription off the chromosome, dysregulation ofthe stringent response, with reduced bacterial fitness in challenging microenviron-ments like blood, may result.

Alterations to amino acid metabolism are consistently among the reported SR-mediated effects (39, 63). It is therefore not surprising that conserved changes in the

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expression of arginine deiminase pathway genes were observed from transcriptomicanalysis of GBS strains A909 and 10/84 grown under SR and control conditions.Alterations in arginine deiminase pathway expression have also been reported in GBSand Streptococcus pyogenes in response to human blood or serum, with available datasuggesting that exposure to blood triggers dynamic arginine deiminase pathwayexpression changes and that, in the case of S. pyogenes, some of those changes aremediated by the SR (35, 47, 64, 65). �HC expression has also been shown to promoteGBS survival in human blood and in a murine sepsis model. The likely mechanism isthrough resistance to phagocytic killing (4).

The change in �HC expression by GBS during SR growth was surprising, however.While others have identified a role of the arginine deiminase pathway in controlling thevirulence of GBS and related species (35, 66, 67), we are the first to connect SRactivation, arginine deiminase pathway expression changes, and �HC regulation. Ourproposed model of how intracellular arginine availability regulates GBS virulencethrough �HC expression is presented in Fig. 7. At this point, we do not know whetherarginine feeds directly into �HC biosynthesis or whether it acts in a moonlightingcapacity as a signaling factor (68). Given that the proposed �HC biosynthetic pathwaydoes not include arginine, we suspect the latter mechanism (69). We note that theexpression of multiple cyl genes was upregulated in our SR-versus-control transcrip-tomic analysis, as well as in the comparison between baseline 10/84 and A909 geneexpression levels. The cyl operon is known to be regulated by the CovR/S two-component system (70), but its regulation could also be affected by arginine. This isanother potential topic for future study.

This work suggests multiple topics for translational investigations of candidate drugs orvaccines. Small molecule inhibitors of the SR have been developed as antimicrobials andhave shown some efficacy in preclinical trials, although there are concerns about off-targetside effects (71–73). There have been no studies of SR inhibitors for the prevention ortreatment of GBS infection. Although canavanine effectively reduced GBS �HC expressionin this study, its toxicity profile limits its potential as an antimicrobial (74). However, other,nontoxic arginine antagonists might prove efficacious against GBS infection. Finally, the setof genes that did not meet the strict threshold for being CE in our study but which haddecreased transposon insertions suggestive of near essentiality (Data Set S1 in the supple-mental material) represents a source of hypotheses about potential drug targets.

In summary, we have presented evidence of a previously unknown virulence pathwayin GBS, by which SR activation triggers changes in intracellular arginine homeostasis.Increased arginine availability, in turn, leads to upregulation of the cytotoxin �HC. We

FIG 7 Proposed model of the interaction between SR activation, arginine availability, �HC expression,and cytotoxicity. Based on our data, we believe that GBS arginine availability, which can be functionallylimited by canavanine, is a mediator of �HC expression and cytotoxicity. This same arginine-mediatedmechanism is activated by the SR, which decreases arginine deiminase activity and also enhances GBSsurvival in whole blood.

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believe this work has implications for GBS pathogenesis and, potentially, drug develop-ment.

MATERIALS AND METHODSBacterial strains and growth conditions. GBS strains A909 (serotype Ia, sequence type 7) and 10/84

(serotype V, sequence type 26) and their derivatives were grown at 37°C under stationary conditions inTSB (product number DF0370-17-3; Fisher Scientific) or new Granada medium (45), supplemented with5 �g/ml Erm and 5 �g/ml chloramphenicol (Cm) as needed for selection. For the ppGpp detectionexperiments, GBS was grown on Luria broth (LB) plates to avoid exposing the bacteria to the higherlevels of unlabeled phosphorus present in TSB. Escherichia coli was grown at 37°C with shaking in LBmedium supplemented with 200 �g/ml Erm and 20 �g/ml Cm as needed for selection.

Human whole blood collection. Volunteers for phlebotomy were enrolled after providing informedconsent under an active, approved New York University IRB protocol (i15-01305). Antecubital fossaphlebotomy was performed on three healthy adults. Filter-sterilized aqueous heparin sodium salt wasadded to a final concentration of 2.7 mU/ml. The heparinized blood was used immediately for GBScoincubation.

Mutant library coincubation with human whole blood. The Tn-seq-compatible transposon mutantlibraries A2, A5, and A7, which are in an A909 background and whose generation has been describedpreviously, were thawed from 2-ml amounts of glycerol stocks stored at �80°C and combined (19). Thepooled library was washed three times in 10 ml phosphate-buffered saline (PBS) and then suspended in3 ml PBS. The 3-ml resuspended library was used to seed 100 ml of TSB Erm, which was grown overnightat 37°C.

The next morning, 20 ml of the overnight library outgrowth culture was used to seed 500 ml of freshTSB Erm, which was grown to an optical density at 600 nm (OD600) of 0.8 (mid-log phase). Sixty milliliterswas removed, pelleted by centrifugation, washed once with 20 ml PBS, and then pelleted again andresuspended in 2.5 ml PBS. Serial dilutions of this preparation were plated to determine the initialinoculum, and then 400 �l was used to seed each of five freshly drawn blood samples to 1.5 (107) CFU/ml.A separate 100-ml control outgrowth culture of TSB Erm was seeded with 400 �l and kept at 37°C.

The seeded blood samples were incubated on a rotary mixer at 37°C for 6 h. After coincubation, eachblood sample and the control culture were serially diluted and plated to determine colony counts. Fivehundred-microliter amounts of each blood sample and the control culture were used to seed outgrowthcultures in 500 ml prewarmed TSB Erm, which were grown under stationary conditions at 37°C overnight.

Tn-seq DNA preparation and sequencing. Thirty milliliters from each coincubation outgrowthculture was pelleted and resuspended in 150 �l PBS. Genomic DNA was purified using the MoBioPowerSoil kit according to the manufacturer’s instructions. The DNA yields were 15 to 46 ng/�l asmeasured by spectrophotometry.

Purified DNA was digested with MmeI and ligated to barcoded adapters as described previously (21).Data Set S3 in the supplemental material lists the barcodes used for each of the five experimentalsamples and the library outgrowth control. Selective PCR amplification of transposon-genome junctionswas performed using primers Illumina PCR Tn F and Illumina PCR Adapt R. PCR was limited to 20 to 26cycles in order to remain in the linear phase of template amplification. Following PCR and agarose gelelectrophoresis, the expected 189-bp band was excised and gel extracted using the Qiagen QIAquick kit.Purified samples were assessed on an Agilent Bioanalyzer before sequencing. Amplicon samples weremultiplexed and sequenced on a 150-nt paired-end run of the Illumina HiSeq 4000 platform, with a targetnumber of reads per library of �50 million. Demultiplexing and read binning were performed using theopen source tool FastqMultx (75).

Determination of conditionally essential genes. Demultiplexed Illumina reads were trimmed offlanking adapter and transposon sequences using the open source tool Cutadapt (76). GBS-specificsequences that were �12 nt or �25 nt were discarded. The remaining sequences were aligned to theA909 genome (GenBank accession number NC_007432) using BowTie2 (77).

The resultant BAM files were uploaded to ESSENTIALS and analyzed with the following parameters:Loess genomic position bias off, read count normalization with TMM (trimmed mean of M values), datadispersion estimation with qCML (quantile-adjusted conditional maximum likelihood), and tagwisemodeling of variance with amount of smoothing set to 5. The expected versus experimental insertiondensity fold change cutoff generated by ESSENTIALS was used to separate CE from nonessential genes.The TA site insertion tallies for the data in Fig. 1 were generated using ARTIST (78).

Generation of GBS mutants. The vector insertion mutant GBS ΔrelA was generated using thetemperature-sensitive shuttle vector pHY304 as previously described (79), with the following details.pHY304 was linearized with SpeI and gel extracted. A 500-bp internal fragment of the A909 relA genewith terminal overhangs complementary to the pHY304 free ends, generated by SpeI digestion, wasamplified using PCR primers relA_intF and relA_intR. The fragment was gel extracted and combined withthe purified digest of pHY304 in a Gibson assembly reaction mixture to make pHY304:relA_frag, whichwas subsequently used to transform chemically competent E. coli DH5� with Erm selection at 28°C.Transformants were confirmed by PCR using pHY304 primers that flank the intended insertion site(pHY304_mcsF and PHY304_mcsR).

Miniprepped plasmid samples were then used to transform electrocompetent A909 with TSB Ermselection at 28°C as described previously (19). We used PCR with pHY304_mcsF and PHY304_mcsR toconfirm that the resultant colonies carried the intended plasmid. Single-cross vector insertion mutantswere generated by transitioning liquid cultures of transformed A909 from 28°C to 37°C at early to mid-loggrowth while under Erm selection. Correct insertion of the plasmid in the relA gene was confirmed with

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PCR using relA_outsideF and pHY304_mcsF, followed by Sanger sequencing with pHY304_mcsF toconfirm that the expected pHY304-A909 genome junction had the predicted sequence (data not shown).

A909 ΔcodY and ΔcpsE were generated using analogous procedures with the corresponding PCRprimers listed in Data Set S4. For A909 ΔcpsE, successful disruption of the cpsE gene was functionallyconfirmed based on a negative GBS serotype Ia latex agglutination test rather than PCR (data not shown).

To generate the complemented ΔrelA�pDC123:relA strain, the shuttle vector pDC123 was linearizedwith BamHI. The A909 relA gene and its promoter were amplified with the primers relA_compF andrelA_compR, which have appropriate overhanging sequences complementary to pDC123. The plasmidand insert were ligated with Gibson assembly and transformed into E. coli DH5� with Cm selection. AfterPCR confirmation of successful transformation, the plasmid was miniprepped and used to transformelectrocompetent A909 ΔrelA with selection on TSB agar Erm plus Cm. Successful transformation of theclone used in experiments was confirmed by PCR.

A909 ΔcylE was a generous gift from Victor Nizet. This strain features allelic replacement of the cylEgene with the Cm resistance cassette cat, generated using established methods (79). It exhibits weak Cmresistance, so it was grown on TSB agar without selection.

Thin-layer chromatography for ppGpp detection. The method described by Cashel for (p)ppGppdetection from E. coli was adapted as follows (80). A909 WT, ΔrelA, and ΔcodY were grown overnight onLB plates with appropriate selection. Individual colonies were scraped from the agar and resuspended in5 ml MOPS (morpholinepropanesulfonic acid) minimal medium without supplemental phosphorus orserine to achieve an OD600 of 0.9. Each bacterial suspension was then divided into two 65-�l aliquots, towhich 10 �l 32P was added for a final concentration of �100 mCi/ml. For the SR activation conditions,SHX in MOPS was added to a final concentration of 1 mg/ml; control samples were spiked with an equalvolume of MOPS without SHX. After 30 min, one volume of 13 M formic acid was added to the samples,which were then subjected to three sequential freeze-thaw cycles. The bacterial debris was pelleted bycentrifugation, and the supernatants spotted to polyethyleneimine cellulose-coated thin-layer chroma-tography plates, where they were allowed to dry. The plates were run in covered beakers with 1.5 MKH2PO4. Once this buffer was near the top of the plate, the plate was dried and exposed to autoradiog-raphy film overnight, which was then developed and photographed.

Blood coincubation with KO strains. Fifty-milliliter cultures of GBS were grown in TSB with appro-priate selection overnight. The cultures were normalized to an OD600 of 1.0, and then 30-ml aliquots werepelleted and washed once with 10 ml PBS. After repeat centrifugation of the washed bacteria, the pelletwas resuspended in 2.5 ml of PBS, and serial dilutions of this suspension were plated to determine theinput inoculum.

Four hundred microliters of the bacterial suspension was injected into 4 ml of freshly drawn,heparinized human whole blood, which was maintained on a rotary mixer for 4 h, at which point serialdilutions were plated on TS agar for colony counts. Fractional survival for each sample was calculated asthe output CFU concentration divided by the starting CFU concentration.

Stringent response RNA-seq. Amounts of 150 �l of overnight cultures of A909 and 10/84 in TSBwere used to seed 45 ml of prewarmed, filter-sterilized TSB with or without 1 mg/ml SHX. Each conditionwas tested in triplicate. The cultures were grown under stationary conditions until they reached an OD600

of 0.6. Whole RNA was then purified from 13-ml samples using the Ambion RiboPure bacterial kitaccording to the manufacturer’s instructions. RNA samples were treated with DNase twice, for 1-hintervals, with enzyme inactivation between treatments. Samples were analyzed on an Agilent bioana-lyzer, which demonstrated a mean RNA concentration of 162 ng/�l and a mean RNA integrity number(RIN) of 9.4. rRNA was removed with Illumina Ribo-Zero treatment according to the manufacturer’sinstructions. Enriched mRNA was fragmented and used for synthesis of strand-specific cDNA using theNEBNext Ultra directional RNA library preparation kit (NEB catalog number E7420L). The DNA waspurified between enzymatic reactions, and size selection of the library performed with AMPure SpriSelectBeads (Beckman Coulter Genomics). The titers and sizes of the libraries were assessed on the LabChip GX(PerkinElmer) and with the library quantification kit for Illumina (Kapa Biosciences). Libraries weresequenced on the Illumina HiSeq 2500 platform using 125-nt paired-end reads, with a target of 40 millionreads per library. Following demultiplexing, sequences were aligned to the reference A909 (GenBankaccession number NC_007432) and 10/84 (GenBank accession number NZ_CP006910.1) genomes usingBowtie version 0.12.9. Genes with a significant treatment effect (up- or downregulation in SHX) weredetermined with DESeq version 1.10.1 (with the following cutoffs: P value, �0.05; read count percentile,�0.25; and fold change, �2).

For A909 versus 10/84 transcriptome comparisons, RNA-seq results from the two strains grown underSR and non-SR conditions were normalized for read numbers. Orthologous genes shared by the twostrains were identified using the CloVR-Comparative pipeline (81, 82) and Sybil (83). Normalized readcounts for orthologs were compared directly using in-house scripts.

Hemolysis assays. Fifty-milliliter cultures of GBS were grown in TSB or new Granada medium withappropriate selection and the additives indicated in Fig. 3 and 6, after confirming that none of theadditives changed the broth pH from �7.0. Additive concentrations were 1 mg/ml (0.83 �M) or 0.5mg/ml (0.42 �M) SHX, 0.83 �M L-serine, 10 mM arginine, and 1 mM canavanine. The cultures werenormalized to an OD600 of 1.0, and then 30-ml aliquots were pelleted and washed once with 10 ml PBS.After repeat centrifugation of the washed bacteria, the pellet was resuspended in 1 ml of PBS, and serialdilutions of this suspension were plated to determine the CFU concentration (mean � 2 � 108 CFU/ml).This sample was diluted 1:50 and combined 1:1 with a preparation of 1% washed, packed humanerythrocytes in Hanks’ buffered saline solution (HBSS). Coincubation was for 90 min. Hemolysis rates weredetermined by spectrophotometric measurement of free hemoglobin using established methods (84).

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For additive-only negative controls, the same protocol was followed, replacing GBS with sterile additivedissolved in PBS to the concentrations indicated in Fig. 3 and 6. Hemolysis (OD) is reported as thepercentage relative to the result for treatment with a 1% solution of Triton X-100 in HBSS.

LDH assay. Human cervical epithelial cell cultures (HeLa, ATCC CCL2) were grown to confluence in24-well plates at 37°C and 5% CO2 in Eagle’s minimum essential medium with supplemental fetal bovineserum (FBS), sodium pyruvate, and ciprofloxacin following standard protocols. GBS strains were grownin TSB with appropriate Erm selection for KO strains. At an OD600 of 0.6 (mid-log phase), bacteria werepelleted, resuspended in RPMI medium, and adjusted to achieve a multiplicity of infection of 10. Serialdilutions were plated on TS agar to confirm the correct CFU concentration (data not shown). Prior tocoincubation, the HeLa cells were washed three times with RPMI medium without added supplements(15 min per wash). Amounts of 500 �l of GBS samples were added to the experimental wells. RPMImedium alone served as the negative control, while 1% Triton X-100 in RPMI medium was the positivecontrol. The cell plate was spun at 200 relative centrifugal force (RCF) for 2 min and then returned to 37°Cand 5% CO2 for 4 h. Following coincubation, 300 �l supernatant from each well was analyzed for LDHconcentration using the Roche cytotoxicity detection kit (catalog number 04744926001), following themanufacturer’s instructions.

Statistics and data visualization. t tests with Bonferroni corrections for multiple comparisons wereperformed using GraphPad Prism 7.0. The Tn-seq Circos plot (Fig. 1) was generated using Circos 0.67-7(26). Heat map data for Fig. 5 were generated using HeatmapGenerator 5.0 (85). Gene set enrichmentanalysis for the data in Table 2 was performed with Genome2D (52).

Availability of data. RNA-seq reads are available under GEO accession number GSE98398. Tn-seqreads are available under BioProject accession number PRJNA416503.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00612-17.

SUPPLEMENTAL FILE 1, XLSX file, 0.2 MB.SUPPLEMENTAL FILE 2, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 3, XLSX file, 0.1 MB.SUPPLEMENTAL FILE 4, XLSX file, 0.3 MB.SUPPLEMENTAL FILE 5, PDF file, 4.0 MB.

ACKNOWLEDGMENTSWe are grateful to Victor Nizet for the kind gift of strain A909 ΔcylE. Robert

Washburn provided valuable assistance with thin-layer chromatography for GBS ppGppdetection. This work utilized computing resources at the High-Performance ComputingFacility at NYU Langone Medical Center.

This work was supported by NIH/NIAID grants number R01 AI092743, R33 AI098654,and R21 AI111020 to A.J.R., John M. Driscoll, Jr., M.D. Children’s Fund (ColumbiaUniversity Department of Pediatrics), and the Pediatric Scientist Development Program(NIH/NICHD grant number K12 HD000850) to T.A.H.

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