HOST-ASSOCIATED MICROBIAL COMMUNITIES crossmsequences in GBS (Streptococcus agalactiae) and S....

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A Vaginal Tract Signal Detected by the Group B Streptococcus SaeRS System Elicits Transcriptomic Changes and Enhances Murine Colonization Laura C. C. Cook, a Hong Hu, b Mark Maienschein-Cline, b Michael J. Federle a a Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USA b Core for Research Informatics, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois, USA ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) can colonize the human vaginal tract, leading to both superficial and serious infections in adults and neonates. To study bacterial colonization of the reproductive tract in a mammalian system, we employed a murine vaginal carriage model. Using transcriptome se- quencing (RNA-Seq), the transcriptome of GBS growing in vivo during vaginal car- riage was determined. Over one-quarter of the genes in GBS were found to be dif- ferentially regulated during in vivo colonization compared to laboratory cultures. A two-component system (TCS) homologous to the staphylococcal virulence regulator SaeRS was identified as being upregulated in vivo. One of the SaeRS targets, pbsP,a proposed GBS vaccine candidate, is shown to be important for colonization of the vaginal tract. A component of vaginal lavage fluid acts as a signal to turn on pbsP expression via SaeRS. These data demonstrate the ability to quantify RNA expression directly from the murine vaginal tract and identify novel genes involved in vaginal colonization by GBS. They also provide more information about the regulation of an important virulence and colonization factor of GBS, pbsP, by the TCS SaeRS. KEYWORDS two-component signal transduction, Streptococcus agalactiae, RNA-Seq, vaginal colonization S treptococcus agalactiae (group B streptococcus [GBS]) is an important human pathogen most known for its ability to cause deadly neonatal infections. The primary risk factor to newborns is maternal colonization with GBS in the genitourinary tract (1). In 2010, the CDC revised guidelines to prevent these infections, calling for universal screening of all pregnant women between 35 and 37 weeks of gestation (2). Treatment of GBS in pregnant females involves intrapartum intravenous antibiotics, which has decreased the incidence of neonatal GBS sepsis but has not affected rates of late-onset disease in newborns over 1 week old (3). Maternal intrapartum prophylactic antibiotics have also been shown to have deleterious effects on the intestinal flora of newborns, including a decrease in the frequency of beneficial bifidobacterial species (4). A GBS vaccine has been proposed as a more advantageous strategy to prevent maternal colonization rather than treating infection once it is detected. Development of a GBS vaccine or other anticolonization strategies requires a more thorough under- standing of the genetic profiles of GBS during vaginal carriage. The vaginal environment is made up of a complex and dynamic microbial commu- nity. Environmental stressors on GBS colonizing the vaginal tract include changes during the menstrual cycle in pH, the normal colonizing flora, and host innate immune factors, such as interleukin-17 (IL-17) (5–7). The molecular components necessary for vaginal colonization by GBS have been the subject of study in recent years, with Received 20 October 2017 Returned for modification 8 December 2017 Accepted 19 January 2018 Accepted manuscript posted online 29 January 2018 Citation Cook LCC, Hu H, Maienschein-Cline M, Federle MJ. 2018. A vaginal tract signal detected by the group B streptococcus SaeRS system elicits transcriptomic changes and enhances murine colonization. Infect Immun 86:e00762-17. https://doi.org/10.1128/IAI .00762-17. Editor Nancy E. Freitag, University of Illinois at Chicago Copyright © 2018 American Society for Microbiology. All Rights Reserved. Address correspondence to Laura C. C. Cook, [email protected]. HOST-ASSOCIATED MICROBIAL COMMUNITIES crossm April 2018 Volume 86 Issue 4 e00762-17 iai.asm.org 1 Infection and Immunity on May 21, 2021 by guest http://iai.asm.org/ Downloaded from

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A Vaginal Tract Signal Detected by the Group B StreptococcusSaeRS System Elicits Transcriptomic Changes and EnhancesMurine Colonization

Laura C. C. Cook,a Hong Hu,b Mark Maienschein-Cline,b Michael J. Federlea

aDepartment of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois,USA

bCore for Research Informatics, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois,USA

ABSTRACT Streptococcus agalactiae (group B streptococcus [GBS]) can colonize thehuman vaginal tract, leading to both superficial and serious infections in adults andneonates. To study bacterial colonization of the reproductive tract in a mammaliansystem, we employed a murine vaginal carriage model. Using transcriptome se-quencing (RNA-Seq), the transcriptome of GBS growing in vivo during vaginal car-riage was determined. Over one-quarter of the genes in GBS were found to be dif-ferentially regulated during in vivo colonization compared to laboratory cultures. Atwo-component system (TCS) homologous to the staphylococcal virulence regulatorSaeRS was identified as being upregulated in vivo. One of the SaeRS targets, pbsP, aproposed GBS vaccine candidate, is shown to be important for colonization of thevaginal tract. A component of vaginal lavage fluid acts as a signal to turn on pbsPexpression via SaeRS. These data demonstrate the ability to quantify RNA expressiondirectly from the murine vaginal tract and identify novel genes involved in vaginalcolonization by GBS. They also provide more information about the regulation of animportant virulence and colonization factor of GBS, pbsP, by the TCS SaeRS.

KEYWORDS two-component signal transduction, Streptococcus agalactiae, RNA-Seq,vaginal colonization

Streptococcus agalactiae (group B streptococcus [GBS]) is an important humanpathogen most known for its ability to cause deadly neonatal infections. The

primary risk factor to newborns is maternal colonization with GBS in the genitourinarytract (1). In 2010, the CDC revised guidelines to prevent these infections, calling foruniversal screening of all pregnant women between 35 and 37 weeks of gestation (2).Treatment of GBS in pregnant females involves intrapartum intravenous antibiotics,which has decreased the incidence of neonatal GBS sepsis but has not affected rates oflate-onset disease in newborns over 1 week old (3). Maternal intrapartum prophylacticantibiotics have also been shown to have deleterious effects on the intestinal flora ofnewborns, including a decrease in the frequency of beneficial bifidobacterial species(4). A GBS vaccine has been proposed as a more advantageous strategy to preventmaternal colonization rather than treating infection once it is detected. Developmentof a GBS vaccine or other anticolonization strategies requires a more thorough under-standing of the genetic profiles of GBS during vaginal carriage.

The vaginal environment is made up of a complex and dynamic microbial commu-nity. Environmental stressors on GBS colonizing the vaginal tract include changesduring the menstrual cycle in pH, the normal colonizing flora, and host innate immunefactors, such as interleukin-17 (IL-17) (5–7). The molecular components necessary forvaginal colonization by GBS have been the subject of study in recent years, with

Received 20 October 2017 Returned formodification 8 December 2017 Accepted 19January 2018

Accepted manuscript posted online 29January 2018

Citation Cook LCC, Hu H, Maienschein-Cline M,Federle MJ. 2018. A vaginal tract signaldetected by the group B streptococcus SaeRSsystem elicits transcriptomic changes andenhances murine colonization. Infect Immun86:e00762-17. https://doi.org/10.1128/IAI.00762-17.

Editor Nancy E. Freitag, University of Illinois atChicago

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Laura C. C. Cook,[email protected].

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individual adhesins such as serine-rich-repeat proteins and regulators being associatedwith increased vaginal carriage in murine models (5, 8–10). Here we determine, for thefirst time, the full transcriptional profile of GBS strain A909 during murine vaginalcolonization compared to laboratory culture conditions. Transcriptome sequencing(RNA-Seq) studies described here show that numerous global changes occur in bacte-rial transcription during vaginal colonization, including widespread metabolic shifts,differential expression of numerous transcriptional regulators, and the upregulation ofmany putative adherence factors. These data will be invaluable for future studiesexamining GBS colonization factors as well as indicating potential vaccine targets andtherapeutics aimed at preventing GBS vaginal colonization in women.

Our findings show that a two-component system (TCS) homologous to the SaeRSvirulence-associated TCS in Staphylococcus aureus was highly upregulated in GBSduring vaginal colonization. As a canonical TCS in S. aureus, SaeS serves as a sensorhistidine kinase, modulating the phosphorylation state of its cognate response regu-lator, SaeR, in response to environmental signals. Phosphorylated SaeR binds DNA andacts as a direct transcriptional regulator of a wide variety of virulence factors in S.aureus, including alpha-hemolysin and coagulase, among other important exoproteins,and the role of the SaeRS TCS in S. aureus virulence has been demonstrated bynumerous studies (11–15). Because genes of the S. aureus SaeRS regulon have noapparent homologs in GBS, the role that SaeRS plays in gene regulation and the signalthat it senses in GBS is unknown. Here, we identify genes controlled by the SaeRS TCSsystem during growth in a murine model of vaginal colonization and demonstrate thatat least one of these genes is an important factor in GBS colonization or survival in thevaginal tract. Finally, we show that a signal present in vaginal lavage (VL) fluid frommice is sufficient to induce SaeRS-dependent gene expression.

RESULTSTranscriptomic analysis of GBS during vaginal colonization signals a shift in

genetic programming. The pathogen GBS is most recognized as a vaginal colonizer,so we used RNA-Seq to measure genome-wide mRNA levels during growth in a murinevaginal colonization model and compared them to those occurring during growthunder laboratory culturing conditions. GBS cultures grown statically at 37°C in achemically defined medium (CDM) were compared to bacteria collected from thevaginal tract 48 h following initial inoculation. Approximately one-third of the entiregenome of strain A909, 731 genes, were identified as being differentially expressed,with a false-discovery rate (q value) of �0.05, in the vaginal tract compared to genesin log-phase growth in CDM. Of these, 630 were differentially regulated �2-fold(Fig. 1A; see also Table S1 in the supplemental material). Pathway enrichment againstKEGG metabolic pathways was performed using Kyoto Encyclopedia of Genes andGenomes (KEGG) orthologous (KO) group assignments; metabolic pathways that aredifferentially expressed during vaginal growth are shown in Fig. 1B.

Phosphotransferase systems (PTSs) are almost universally highly upregulated duringin vivo colonization (Table 1 and Fig. 1B), with some systems being upregulated�500-fold. PTSs are one important method that bacteria use to sense carbon sourceavailability and transport nutrients into the cell.

The fab operon, which regulates fatty acid biosynthesis, was highly downregulatedduring vaginal colonization (Table 1 and Fig. 1B). Lowered expression of genes in thisoperon is thought to increase the amount of long-chain unsaturated fatty acids, whichcan be protective during growth at a low pH (16, 17). As the human vaginal tract isnormally at pH ranges between 3.8 and 4.5, an overall low expression of the fab operonmay provide GBS a competitive advantage in the acidic environment.

Differential regulation of two-component systems during growth in vivo. Arecent study defined 21 two-component systems highly conserved in GBS (18). A909contains 20 of these TCSs, and in our analysis, three complete TCSs were differentiallyexpressed between conditions of lab culturing and host colonization (Table 1), with anadditional three histidine kinases being differentially expressed without differential

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expression of the cognate response regulator. All but one of these differentiallyexpressed TCSs were upregulated during in vivo growth.

One of the most highly upregulated TCSs is SAK_0467/0468. This TCS is homologousto the well-characterized SaeR (48% identical) and SaeS (34% identical) TCSs found inStaphylococcus aureus. In S. aureus, SaeRS regulates numerous virulence factors, suchas alpha-hemolysin, lipase protein A, and adhesins, among others (13, 15, 19). Theorganization of the TCS locus in S. aureus is different from a canonical TCS in that itcontains two additional genes (saeP and saeQ) immediately upstream and overlappingwith the saeR gene (Fig. 2A). In S. aureus, saeP and saeQ are required to activate SaeSphosphatase activity, allowing the removal of the activating phosphate on SaeR,effectively recycling it and turning off SaeR activity (20). Alignment of the SaeR genesequences in GBS (Streptococcus agalactiae) and S. aureus is shown in Fig. 2B, withimportant residues highlighted. In the GBS homologous system, saeP and saeQ are notpresent, and in their place is a large gene, sak_0466, encoding a putative cell wallsurface anchor family protein recently named plasminogen binding surface protein(PbsP). PbsP was shown to be important for both plasminogen binding and dissemi-nation of GBS during invasive disease in a different strain of GBS, NEM316 (21).

The genetic pathways controlled by SaeRS are different in S. aureus and in GBS, asthe S. aureus regulon, which is well documented, comprises genes that have nohomologs in GBS. Because SaeRS is upregulated during vaginal growth and is likely toregulate a distinct set of genes, we examined transcriptomic changes between A909and an isogenic ΔsaeR strain during log-phase growth in two different laboratory media(chemically defined medium [CDM] and Todd-Hewitt broth supplemented with yeastextract [THY]) and during colonization of the vaginal tract.

The SaeR-regulated transcriptome differs greatly between in vitro and in vivoconditions. Transcriptomic data indicated that SaeRS regulates, either directly orindirectly, a large portion of the genome of GBS under certain conditions. A q value of0.05 and a cutoff of �2-fold change were the criteria that we used to define differen-tially expressed genes (DEGs). Compared to the wild-type (WT) strain, during log-phase

FIG 1 Transcriptional changes during in vivo growth. (A) Volcano plot of transcriptomic changes in liquid versus mouse. Log2 changes in gene expression wereplotted versus the �log10 false discovery rate (FDR). Genes shown in red have an FDR of �0.05 and a fold change of �2. Three genes discussed in the textare labeled with gene numbers. (B) Pathways statistically overrepresented during growth in vivo.

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TABLE 1 Gene categories differentially expressed during vaginal colonization

Gene category and no.(strain A909) Gene name Descriptiona

Fold change in vaginalcolonization

Phosphotransferasesystems

sak_0257 Trehalose-specific IIBCA component �2.700sak_0261 Sugar-specific IIC component 5.300sak_0354 IIBC component �20.520sak_0398 IIA component, lactose/cellobiose family 31.250sak_0399 IIB component, lactose/cellobiose family 18.700sak_0400 IIC component, lactose/cellobiose family 26.070sak_0524 Galactitol-specific IIA component 31.890sak_0525 Galactitol-specific IIB component 53.170sak_0526 Glucose-specific IIABC component 100.730sak_0528 Galactitol-specific IIA component 52.970sak_0529 Galactitol-specific IIC component 68.580sak_0530 Galactitol-specific IIB component 140.590sak_1377 Fructose-specific IIABC component 7.730sak_1702 Sucrose-specific IIABC component 8.400sak_1759 Fructose-specific, IIC component 5.800sak_1825 Galactitol-specific IIC component 23.700sak_1833 IIA component 684.810sak_1834 IIB component, lactose/cellobiose family 948.230sak_1835 Sugar-specific IIC component 88.170sak_1893 IIC component 84.130sak_1894 IIB component 76.440sak_1895 IIA component 65.180sak_1908 IID component, mannose/fructose/sorbose family 209.060sak_1909 IIC component, mannose/fructose/sorbose family 123.500sak_1910 IIB component, mannose/fructose/sorbose family 88.780sak_1911 IIA component, mannose/fructose/sorbose family 26.670sak_1920 Glucose-specific IIABC component 16.460

ABC transporterssak_0166 rbsB Ribose ABC transporter, ribose-binding protein 102.631sak_0167 rbsC Ribose ABC transporter, permease protein 69.165sak_0168 rbsA Ribose ABC transporter, ATP-binding protein 61.861sak_0169 rbsD Ribose ABC transporter protein RbsD 31.775sak_0207 oppD Oligopeptide ABC transporter, permease protein �2.191sak_0208 Oligopeptide ABC transporter, permease protein OppC �2.085sak_0209 oppD Oligopeptide ABC transporter, ATP-binding protein �2.245sak_0210 oppF Oligopeptide ABC transporter, ATP-binding protein �2.498sak_0302 QAT family ABC transporter, permease �2.163sak_0303 QAT family ABC transporter, ATP-binding �2.255sak_0472 BioY family protein 4.521sak_0561 ABC transporter, ATP-binding/permease protein �4.218sak_0562 ABC transporter, ATP-binding/permease protein �4.435sak_0795 cylA ABC transporter, ATP-binding protein CylA 2.903sak_0796 cylB ABC transporter, permease protein CylB 3.487sak_0899 Amino acid ABC transporter, ATP-binding protein, putative �5.711sak_0900 Amino acid ABC transporter, permease protein, putative �2.611sak_0901 ABC transporter, substrate-binding protein �2.416sak_1074 ABC transporter, substrate-binding protein �8.399sak_1103 Iron chelate uptake ABC transporter, ATP-binding protein 3.031sak_1104 Iron chelate uptake ABC transporter, permease protein 3.352sak_1105 Iron chelate uptake ABC transporter, permease protein 2.848sak_1426 fhuD Ferrichrome ABC transporter, ferrichrome-binding protein 3.423sak_1427 fhuB Ferrichrome ABC transporter, permease protein 4.479sak_1476 Cyclodextrin ABC transporter, permease protein 4.363sak_1477 Cyclodextrin ABC transporter, permease protein 4.452sak_1538 nikE Nickel ABC transporter, ATP-binding protein 38.719sak_1539 nikD Nickel ABC transporter, ATP-binding protein 44.320sak_1540 nikC Nickel ABC transporter, permease protein 68.911sak_1541 nikB Nickel ABC transporter, permease protein 78.738sak_1542 nikA Nickel ABC transporter, nickel-binding protein 53.693sak_1554 mtsC Metal ABC transporter, permease protein 47.051sak_1555 mtsB Metal ABC transporter, ATP-binding protein 52.891sak_1556 mtsA Metal ABC transporter, metal-binding lipoprotein 36.614

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TABLE 1 (Continued)

Gene category and no.(strain A909) Gene name Descriptiona

Fold change in vaginalcolonization

sak_1594 livF HAAT family ABC transporter, ATP-binding protein �8.380sak_1595 livG HAAT family ABC transporter, ATP-binding protein �7.300sak_1596 livM HAAT family ABC transporter, permease �10.973sak_1597 livH HAAT family ABC transporter, permease �10.450sak_1646 HAAT family ABC transporter, permease �2.663sak_1647 ABC transporter, ATP-binding protein �2.131sak_1747 cydC ABC transporter, ATP-binding protein CydC �2.267sak_1884 msmK Sugar ABC transporter, ATP-binding protein 4.983sak_1925 pstA Phosphate ABC transporter, permease protein PstA 18.213sak_1927 Phosphate ABC transporter, phosphate-binding protein 10.791sak_1966 ABC transporter, ATP-binding protein �3.675sak_2068 Glycine betaine/carnitine/choline ABC transporter, ATP-binding protein �2.434sak_2069 Glycine betaine/carnitine/choline ABC transporter, permease �2.196

Starch and sucrosemetabolism

sak_1472 glgP Glycogen/starch/alpha-glucan phosphorylase 4.686sak_0976 glgB 1,4-Alpha-glucan branching enzyme 3.913sak_0979 glgA Glycogen synthase 5.437sak_1473 malQ 4-Alpha-glucanotransferase 4.785sak_0977 glgC Glucose-1-phosphate adenylyltransferase 5.077sak_0978 glgD Glucose-1-phosphate adenylyltransferase, GlgD subunit 5.519sak_1703 scrB Sucrose-6-phosphate hydrolase 3.684sak_1188 Glycosyl hydrolase, family 1 �2.833sak_0258 Alpha amylase family protein �2.605sak_1155 Phosphoglucomutase/phosphomannomutase family protein 3.877sak_0398 PTS system, IIA component, lactose/cellobiose family 31.254sak_0399 PTS system, IIB component, lactose/cellobiose family 18.697sak_0400 PTS system, IIC component, lactose/cellobiose family 26.073sak_1920 PTS system, glucose-specific IIABC component, putative 16.457sak_1702 PTS system, sucrose-specific IIABC component 8.405sak_0354 PTS system, IIBC component �20.528sak_0257 PTS system, trehalose-specific IIBCA component �2.697

Fatty acid biosynthesisand metabolism

sak_0416 fabM Enoyl-CoA hydratase/isomerase family protein �20.503sak_0417 fabT MarR transcriptional regulator �11.350sak_0418 fabH 3-Oxoacyl-(acyl-carrier-protein) synthase III �11.029sak_0419 acpP Acyl carrier protein �4.304sak_0420 fabK Enoyl-(acyl-carrier-protein) reductase II �19.083sak_0421 fabD Malonyl-CoA-acyl carrier protein transacylase �15.727sak_0422 fabG 3-Oxoacyl-(acyl-carrier-protein) reductase �21.685sak_0423 fabF 3-Oxoacyl-(acyl-carrier-protein) synthase II �10.549sak_0424 accB Acetyl-CoA carboxylase, biotin carboxyl carrier protein �10.054sak_0425 fabZ Beta-hydroxyacyl-(acyl-carrier-protein) dehydratase fabz �12.538sak_0426 accC Acetyl-CoA carboxylase, biotin carboxylase �8.781sak_0427 accD Acetyl-CoA carboxylase, carboxyl transferase, beta subunit �10.492sak_0428 accA Acetyl-CoA carboxylase, carboxyl transferase, alpha subunit �8.249

Fructose and mannosemetabolism

sak_1036 pfk 6-Phosphofructokinase �2.172sak_1888 lacC Tagatose-6-phosphate kinase 102.565sak_0537 galT Galactose-1-phosphate uridylyltransferase 30.080sak_1703 scrB Sucrose-6-phosphate hydrolase 3.680sak_1887 lacD Tagatose 1,6-diphosphate aldolase 104.990sak_0538 galE UDP-glucose 4-epimerase 13.700sak_1889 lacB Galactose-6-phosphate isomerase, LacB subunit 55.168sak_1890 lacA Galactose-6-phosphate isomerase, LacA subunit 123.335sak_1155 Phosphoglucomutase/phosphomannomutase family protein 3.880sak_0524 PTS system, galactitol-specific IIA component, putative 31.886sak_0528 PTS system, galactitol-specific IIA component, putative 52.973sak_0525 PTS system, galactitol-specific IIB component, putative 53.173sak_0530 PTS system, galactitol-specific IIB component, putative 140.593

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growth, 301 genes were differentially expressed in the ΔsaeR strain in CDM and 466genes were differentially expressed in THY. Of those that were different in CDM, 136genes (�45%) were also changed in THY, though a small number were regulated inopposite directions. The results of these experiments are summarized in Fig. 3A and B,and detailed transcriptomic information can be found in Table S4 in the supplementalmaterial. Although many DEGs were observed in liquid culture, the magnitudes ofdifferences were generally low, with only 6/301 genes in CDM and 29/466 genes in THYshowing differences of �10-fold. The clear majority of DEGs were seen to differ only 2-to 5-fold, which may indicate an indirect mechanism of regulation. Despite the largenumber of genes differentially regulated by SaeRS in liquid culture, only three geneswere differentially expressed between the wild-type and the ΔsaeR strains duringgrowth in the vaginal tract (Fig. 3C). In the ΔsaeR mutant, levels of sak_1753 mRNA weremore than 800 times lower than for the wild type during vaginal colonization and levelsof pbsP transcript were more than 200-fold lower. The third gene differentially regu-lated between wild-type and ΔsaeR strains was a predicted small open reading frame(ORF), sak_RS00910, which is located between sak_0183 and sak_0184. This ORF en-codes a putative peptide of 38 amino acids with no predicted function or homolog. Thesak_RS00910 transcript was downregulated a more modest 7.7-fold in the ΔsaeRmutant. Two of these three DEGs, sak_0466 (pbsP) and sak_1753, were among the top10 most highly upregulated genes seen in the wild type during growth in the vaginaltract compared to lab culture (marked in Fig. 1A). pbsP was upregulated over 250-foldduring vaginal colonization compared to growth in CDM, and sak_1753 was the most

TABLE 1 (Continued)

Gene category and no.(strain A909) Gene name Descriptiona

Fold change in vaginalcolonization

sak_0526 PTS system, galactitol-specific IIC component, putative 100.735sak_0529 PTS system, galactitol-specific IIC component 68.575sak_1825 PTS system, IIC component, putative 23.697

Oxidative phosphorylationsak_1750 cydA Cytochrome d ubiquinol oxidase, subunit II �3.030sak_1749 cydB Cytochrome d ubiquinol oxidase, subunit II �2.081sak_1036 pfk 6-Phosphofructokinase �2.170sak_1378 1-Phosphofructokinase, putative 6.540sak_0527 rhaD Rhamnulose-1-phosphate aldolase 83.546sak_0594 Phosphoglucomutase/phosphomannomutase family protein 21.790sak_0981 atpB ATP synthase F0, A subunit �3.005sak_0982 atpF ATP synthase F0, B subunit �2.699sak_0980 atpE ATP synthase F0, C subunit �2.417sak_0983 atpH ATP synthase F1, delta subunit �2.672sak_0985 atpG ATP synthase F1, gamma subunit �2.254sak_1377 PTS system, fructose-specific IIABC component 7.730sak_1759 PTS system, fructose-specific IIC component 5.800sak_1911 PTS system, IIA component, mannose/fructose/sorbose family 26.668sak_1910 PTS system, IIB component, mannose/fructose/sorbose family 88.776sak_1909 PTS system, IIC component, mannose/fructose/sorbose family 123.500sak_1908 PTS system, IID component, mannose/fructose/sorbose family 209.064sak_1751 Pyridine nucleotide-disulfide oxidoreductase family protein �3.130sak_0666 fbp Fructose-1,6-bisphosphatase 3.520

Two-component systemssak_0467 saeR DNA-binding response regulator 8.469sak_0468 saeS Sensor histidine kinase 6.033sak_1880 Sensor histidine kinase, putative 3.625sak_1881 Response regulator 4.978sak_1917 rgfC Histidine kinase 3.879sak_1921 Sensor histidine kinase 2.575sak_2061 DNA-binding response regulator �3.831sak_2062 Sensor histidine kinase �2.506sak_2066 Sensor histidine kinase, putative 53.384

aHAAT, hydrophobic amino acid uptake; CoA, coenzyme A; QAT, quaternary amine uptake.

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highly differentially expressed gene, upregulated over 3,000-fold in vivo (see Table S3in the supplemental material). This evidence indicates that SaeR is responsive toconditions in the vaginal tract and that targets of SaeR regulation could play animportant role during growth in vivo.

Role of PbsP in vaginal colonization. PbsP, a documented surface protein con-taining a sortase-dependent LPXTG motif, was shown to be important in respiratorytract colonization and invasive disease models of infection for GBS and Streptococcuspneumoniae (21–23). Considering that we found pbsP transcript levels to be substan-tially increased during growth of GBS in the vaginal tract, we asked whether removalof the gene would affect its ability to colonize the vaginal tract. We employed a murinemodel of vaginal carriage to look at colonization levels. In this model, mice are injectedwith estrogen to synchronize the estrous cycle 1 day prior to vaginal inoculation withbacteria. Colonization is subsequently measured over time as CFU in vaginal washes.We generated an in-frame deletion mutant, ΔpbsP, and compared its ability with thatof the wild type to colonize and remain within the murine vaginal tract over the courseof several days (Fig. 4). Initial colonization levels 24 h after inoculation were equivalentin WT A909 and the ΔpbsP mutant, but the numbers of viable ΔpbsP bacteria recoveredon day 2 postinoculation were significantly decreased compared to the WT, indicatinga colonization defect in the ΔpbsP mutant (Fig. 4). By later time points, many mice wereno longer colonized and the trending decrease in ΔpbsP mutant colonies was presentbut no longer significant. Addition of a plasmid containing a copy of PbsP under aconstitutive PrecA promoter complemented the colonization defect.

Regulation of pbsP and sak_1753 by SaeRS via a signal present in vaginallavage fluid. The signal recognized by SaeS in GBS is not known, but we hypothesized

FIG 2 saeRS loci and SaeR protein sequences in Streptococcus agalactiae and Staphylococcus aureus. (A)saeRS loci of S. aureus and S. agalactiae, including the surrounding region. (B) SaeR protein sequencealignment between S. agalactiae and S. aureus. Residues marked in yellow were shown to be importantfor DNA binding, and the residue marked in blue is the predicted phosphorylated aspartic acid residuefor the SaeR protein in S. aureus (44).

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FIG 3 The SaeR regulon. (A) Scatter plot shows differentially expressed genes (FDR � 0.05) between theWT and the isogenic ΔsaeR mutant as the log2-fold change in chemically defined medium (CDM) versusrich medium (THY). (B) Venn diagram showing differentially expressed gene overlap in CDM versus THY.(C) Scatter plot showing gene expression in WT A909 versus the isogenic ΔsaeR mutant. The four geneswith an FDR P value of �0.05 are marked in red.

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that a component of the vaginal tract, which may comprise a soluble or cell-associatedfactor not found in laboratory media, may be sensed by SaeS and relayed to SaeR toregulate expression of pbsP and sak_1753. To determine whether such a signal waspresent in material of the vaginal tract, VL fluids from several mice were collected andpooled. For these experiments, mice were not synchronized for estrus, so lavage fluidsfrom 10 mice were pooled to decrease estrous cycle variability. Wild-type or ΔsaeR cellswere then suspended in either VL or phosphate-buffered saline (PBS). Following a 1-hincubation, transcript levels of pbsP and sak_1753 were assessed by quantitativereal-time (RT)-PCR. In WT cells, incubation with VL caused increased transcription ofboth pbsP and sak_1753, approximately 20- to 30-fold over that of levels seen in PBS,corroborating the initial RNA-Seq results described above (Fig. 5A). In ΔsaeR cells, anincrease in transcript amounts for either of these two genes following incubation withVL was not observed (Fig. 5A). These data indicate that the SaeRS system senses acomponent of VL fluid and this signal promotes upregulation of pbsP and sak_1753 viathe response regulator SaeR.

To help determine the nature of the signal sensed by SaeRS, vaginal lavage fluidsamples were treated by heating at 100°C for 30 min, were incubated at 37°C for 30 minwith 1 mg/ml pronase enzyme, or were passed through a 3-kDa filter. Followingtreatments, VL samples were tested for the ability to upregulate expression of pbsP andsak_1753. Lavage fluid treated with heat or pronase no longer retained signaling ability,but filtrates passing through a 3-kDa molecular mass cutoff remained able to induceincreased gene expression, although to a lower level than untreated VL, approximately4- to 8-fold above levels seen in PBS-treated cells (Fig. 5B). These findings indicated thatthe signal is likely to comprise a small, heat-labile peptide of �30 residues.

To determine whether SaeR regulation of pbsP was direct, an electrophoreticmobility shift assay (EMSA) was performed. Because previous studies have demon-strated that SaeR must be phosphorylated in order to bind DNA (24), we attempted tophosphorylate SaeR in vitro using acetyl phosphate. Although we did not observe ashift when using this treatment, we were unable to confirm phosphorylation of SaeR.As it has been shown for several response regulators that replacement of the phos-phorylated aspartic acid residue with a glutamate, which provides an extended sidechain length, can often result in a constitutively active protein (25), we expressed andpurified the SaeR Asp-to-Glu (D53E) variant. Direct binding of the pbsP promoter regionwas observed via an EMSA (Fig. 5C).

DISCUSSION

To persist on mucosal surfaces such as the vaginal tract, bacteria must havemechanisms to adhere to tissues, evade antimicrobial agents, and adapt to changingenvironments and nutrients. Identifying these mechanisms would greatly aid the abilityto develop treatments that target colonizing pathogens. In the case of GBS, the vaginalenvironment has been the primary subject of study, as maternal vaginal colonization

FIG 4 Role of pbsP in vaginal colonization. Colonization levels in CFU/vaginal wash of WT A909 versus theisogenic ΔpbsP mutant on days 1, 2, 3, and 5. Statistical significance was assessed using a nonparametricMann-Whitney test on raw values. *, P � 0.05.

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can result in severe neonatal infections. Although some colonization factors have beendescribed for GBS, an overall picture of the gene profile during in vivo growth is animportant missing piece of the puzzle for a full understanding of GBS carriage andinfection. Here we present in vivo RNA-Seq data quantifying the transcriptome of GBSduring murine vaginal carriage.

The genetic programs governing in vivo growth are shown to be substantiallydifferent from what is seen during growth under laboratory conditions, with overone-quarter of the genome showing differential regulation between these two envi-ronments. Membrane components such as sensor kinases and nutrient transportsystems were generally highly upregulated in the vaginal environment (Fig. 1B). In bothrich and chemically defined media, where nutrients are abundant, it may be unneces-sary for bacteria to induce expression of PTS systems. In the host, on the other hand,the upregulation of phosphotransferase systems may be essential for survival.

FIG 5 Vaginal lavage fluid induces increased gene expression of pbsP and sak_1753 via SaeR. (A) qPCR transcript levels of the indicated genes followingincubation of WT A909 or ΔsaeR mutant cells with vaginal lavage fluid at 37°C for 60 min. (B) Vaginal lavage fluid was treated as indicated with heat, pronase,or filtration prior to incubation with A909. qPCR transcript levels are shown for the indicated genes. All transcript levels were compared to those of ahousekeeping gene, gyrA, and are shown as a ratio (to PBS-treated control levels). Student’s t test was used to analyzed statistical significance *, P � 0.05; **,P � 0.005. (C) EMSA gel showing increased shift of the pbsP promoter DNA probed with increased concentrations of SaeR D53E protein. This shift is no longervisible after addition of 10-fold excess unlabeled target DNA but not with 10-fold excess unlabeled random DNA.

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Metabolic systems were also found to be highly differentially expressed. Of partic-ular importance, the fab fatty acid synthesis operon was substantially downregulatedduring growth in the vaginal tract compared to what was seen under lab culturingconditions. In S. pneumoniae, fatty acid regulation is controlled by the MarR familytranscriptional regulator FabT. Deletion of FabT in S. pneumoniae results in upregulationof all fab genes in the fatty acid synthesis operon besides fabM, indicating that FabTacts as a repressor in S. pneumoniae. The authors proposed that in a FabT-deficientmutant strain, upregulation of the fab genes leads to decreased amounts of membraneunsaturated fatty acids (UFA), which results in sensitivity to acid (17). An increase inlong-chain fatty acids also results in growth benefits in Streptococcus mutans inacidified environments (16). In the vaginal tract, where sensitivity to acid would behighly detrimental, we found that all genes in the fatty acid synthesis operon, includingfabM and fabT, were highly downregulated compared to growth in either CDM or THY,indicating a switch to longer-chain UFA in the vaginal tract than in the laboratorymedia. Downregulation of the fab operon could lead to increased UFA in the mem-brane and protect the bacteria from the acidic vaginal environment. The decrease infabT levels that we observed is surprising if FabT acts as a transcriptional repressorin GBS; however, if FabT is a relatively stable protein, perhaps increased fabTtranscription occurs in the cells during initial stages of colonization, prior to the48-hour sampling time point, leading to an overall transcriptional repression of theentire operon, including fabT.

It is likely that many of the transcriptional differences that we observed in laboratorylog-phase growth and in vivo growth are due to changes in the growth state of theorganisms. It is possible that the in vivo environment would more closely mimic astationary-phase growth in liquid culture. The switch from log-phase to stationary-phase growth in bacteria is often characterized by a decrease in expression of ribo-somal genes, which in this case have been depleted from our input libraries. Arepression in cellular processes associated with cell replication and growth as well astranscription and translation is often observed in the log- to stationary-growth phaseshift (26, 27). A previous study by Sitkiewicz and Musser examined gene expressionchanges in GBS during different growth phases in liquid culture and described acharacteristic gene expression profile during stationary phase, including upregulationof particular metabolic genes and stress response genes as well as differential expres-sion of numerous virulence factors (28). We observe similar patterns of gene expressionfor some of these genes, including high upregulation of the arginine/ornithine car-bamoyltransferases (sak_2063-2065 and sak_2123-2125) and the glp operon (sak_0345-0347) during in vivo growth. Conversely, some of the genes found to be differentiallyexpressed in stationary versus log phase in that study are not regulated similarly underour conditions; fba (sak_0178), gap (sak_1790), pgk (sak_1788), and eno (sak_0713), allfound to be differentially expressed in stationary-phase liquid culture, are not differ-entially expressed in vivo compared to what is seen in log-phase growth. Some genesare even regulated in the opposite manner; gbs1539 was found to be upregulatedduring stationary-phase growth in liquid culture but downregulated in vivo. These datasuggest that although the bacterial cells grown in vivo have certain characteristics ofstationary-phase growth, it would be an oversimplification to assume that the hall-marks of stationary-phase growth are recapitulated in vivo.

Several TCSs were also found to be differentially regulated during in vivo growth.One such system, highly upregulated in the vaginal tract, was identified as a homologof the S. aureus virulence regulatory TCS, SaeRS. Despite a high level of sequencehomology between the SaeRS proteins of S. aureus and GBS, the S. aureus SaeRSregulon is without homologs in GBS and the known SaeR DNA-binding sequence,GTTAAN6GTTAA (24), was not found at promoter regions of genes observed to bedifferentially expressed by SaeR in our RNA-Seq data. Alignment of the SaeR sequencesof S. aureus and GBS shows that the residues associated with DNA binding in S. aureus(H198, R199, R201, and W218) are conserved, with the exception of residue R199 of S.aureus, thought to be less important for binding, which is an alanine in GBS (Fig. 2A).

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However, regions of SaeR flanking the DNA binding helices are not similar and mayimpact the identity of DNA recognition sequences between the orthologs. We havedemonstrated that SaeR D53E is able to bind upstream of pbsP. Further work is underway to identify the exact DNA binding site recognized by SaeR in GBS to more easilydetermine which genes this response regulator directly controls. Although we hypoth-esize that SaeS acts as the sensor kinase to phosphorylate SaeR, as is the case in S.aureus, we cannot rule out the possibility that SaeR acts promiscuously with othersensor kinases to regulate gene expression.

RNA-Seq analysis was undertaken to identify transcripts whose levels were influ-enced by SaeRS under different growth conditions, including during growth in thevaginal tract. SaeRS appears to be involved in the regulation of many GBS genes duringgrowth in liquid culture but accounted for a very small and specific genetic programduring growth in the vaginal tract. Under this condition, SaeR regulated sak_1753, ahypothetical gene with no known homologs, and a putative small peptide-encodinggene, sak_RS00910, which is located between sak_0183 and sak_0184. Importantly,SaeR also regulated sak_0466 (pbsP), which has been shown to be important fordissemination of GBS during invasive disease, and which we show here to be animportant colonization factor for GBS. In liquid culture, the majority of differentiallyexpressed genes were changed by a small degree, generally between 2- and 5-fold,whereas the three DEGs observed during vaginal growth were downregulated 7-fold,�200-fold, and �800-fold. It is possible that the changes seen in liquid culture were theresult of indirect rather than direct regulation in the vaginal tract. It is also possible thatSaeRS is responsible for a vastly different genetic program depending on environmen-tal signals.

Two of the genes regulated by SaeR during vaginal colonization, sak_1753 andsak_0466 (pbsP), were among the most highly differentially expressed genes in thevaginal tract of WT strains compared to those growing in liquid. The role of sak_1753in vaginal carriage is currently being examined, although there are very few knownhomologs and no predicted function for the protein product. PbsP, on the other hand,has recently been identified as a possible GBS vaccine target and was shown to beimportant in hematogenous dissemination during an intravenous infection. It has alsobeen found to be upregulated during growth in human blood (29) and at 40°C (30).PbsP is highly conserved in GBS strains and present in all sequenced GBS clinicalisolates from humans. The role of PbsP in invasive disease was shown in GBS strainNEM316, a clonal complex 23 isolate of capsular serotype III (21). A909, a clonal complex19 isolate with capsular serotype Ia, was used in these studies, indicating the impor-tance of this gene in multiple strains of GBS in vivo.

In GBS, pbsP is located immediately upstream of saeRS and contains a sortase cellwall-anchoring LPXTG motif, and levels of PbsP protein are decreased in an srtA mutantstrain (31). A recent paper examined the role of PbsP in an intravenous model of GBSinfection. In this model, PbsP was important for binding of plasminogen, disseminationto organs such as the kidney, and transendothelial migration into the brain (21). In S.pneumoniae, a homologous gene named pneumococcal adherence and virulencefactor B, pavB (also termed pfbB) was found to be important for adherence to respira-tory epithelial cell lines via fibronectin and plasminogen and for colonization of thenasopharynx (22, 23).

Here we demonstrate that PbsP is also important in maintenance of colonization inthe vaginal tract and that its expression levels, controlled by SaeRS, are highly upregu-lated during incubation with murine vaginal lavage fluid. On day 1 following vaginalinoculation, numbers of A909 and A909ΔpbsP isolates were similar. We hypothesizethat during this 24-hour period, bacteria were not necessarily being actively removedfrom the vaginal tract, and thus the A909ΔpbsP mutants were able to persist in thevaginal environment. Following the day 1 vaginal wash, the bacteria would be requiredto actively adhere to the tissue to remain in the vaginal tract, emphasizing the defectin colonization by the mutant. Thus, its role does not appear to be limited to invasivedisease. In their 2016 article, Buscetta et al. suggest that PbsP plays a role in favoring

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systemic spread of GBS and invasive infection (21). Because their model begins withintravenous (i.v.) infection rather than mucosal carriage, it is possible that they areobserving an unintended side effect of PbsP during systemic infection rather than theprimary role as an adhesin on mucosal surfaces. Previous studies have linked theexpression of pbsP to the master virulence regulator CovRS, as levels of pbsP transcriptand protein are higher in a ΔcovRS strain (21, 32, 33). Direct binding of CovR to thepromoter of pbsP was not observed, and it was postulated that the regulation may beindirect (21). It is possible that SaeRS is being regulated by CovRS, although changes intranscript levels of saeRS were not observed in a ΔcovRS mutant (32). Changes intranscript levels of psbP were altered to a much greater extent in a ΔsaeR mutant thanin the ΔcovRS mutant, and we postulate that the SaeRS system is more likely to be adirect regulator of psbP than CovRS. Our EMSA data also indicate that phosphomimeticSaeR D53E is a direct regulator of pbsP via promoter binding. Based on these data, wepropose that SaeR is phosphorylated by SaeS following interaction with a signalpresent in the vaginal tract and that phosphor-SaeR directly binds upstream of pbsP toincrease gene expression. Alignment of the �200 bp upstream of the ATG start site ofpbsP and sak_1753 shows a surprisingly high degree of sequence similarity (see Fig. S1in the supplemental material). Further work will be needed to characterize the exactDNA binding sequence.

Our studies indicate that the signal is likely a small heat-labile peptide of less than3 kDa. It was shown in S. aureus that subinhibitory concentrations of the humanneutrophil peptide (HNP) �-defensins could induce expression of SaeRS-controlledpromoters (34). This finding led the authors to hypothesize that host-produced anti-microbial peptides could serve as a signal to activate the SaeRS system. It is possiblethat �-defensins or a similar small antimicrobial peptide also serves as a signal for SaeRSin S. agalactiae. The extracellular linker region of SaeS (WFNGHMTLT) was found to beimportant for responding to HNP in S. aureus (35). In S. agalactiae, the linker region hassome similarity (GGLNHMLIET), indicating that the signal sensed by these proteins maybe similar but not identical. Current studies are focusing on identifying the exact natureof the signal sensed by SaeS in VL and determining whether the signal is related toestrous cycle changes. A model of these data is shown in Fig. 6. Signals encountered inthe host environment cause numerous transcriptional changes within the GBS cell. Onetwo-component system, SaeRS, was found to be upregulated during growth in thehost. This system senses a host signal during colonization of the vaginal tract andinduces expression of sak_1753, a gene encoding an unknown hypothetical protein,and pbsP, encoding a putative adhesin and colonization factor, likely via direct binding.We demonstrate the PbsP is important for vaginal colonization. These data provideimportant insight into the molecular programming of GBS during growth and coloni-zation in the vaginal tract.

MATERIALS AND METHODSBacterial strains, media, plasmids, and primers. All strains and plasmids are shown in Table S1,

and the primers used in this study are described in Table S2 in the supplemental material. Escherichia colistrain BH10C (36) was cultivated in Luria-Bertani (LB) medium or on LB agar. When necessary, antibioticswere included at the following concentrations for E. coli propagation: chloramphenicol (Cm), 10 �g ml�1;kanamycin (Kan), 150 �g ml�1; erythromycin (Erm), 100 �g ml�1. All GBS strains used in this study werederived from the clinical isolate A909 (37). GBS were routinely grown in Todd-Hewitt medium (BDBiosciences) supplemented with 2% (wt/vol) yeast extract (Amresco) (THY) or a chemically definedmedium (CDM) modified from that described by van de Rijn and Kessler RE (38, 39). The exactcomposition of CDM and the protocol for preparation have been previously published (39). Plating wasdone on THY agar plates or CHROMagar StrepB agar plates (CHROMagar). When necessary, antibioticswere included at the following concentrations for GBS propagation: chloramphenicol (Cm), 3 �g ml�1;kanamycin (Kan), 150 �g ml�1; erythromycin (Erm), 10 �g ml�1; spectinomycin (Spec), 100 �g ml�1;ampicillin (Amp), 50 �g ml�1.

Mouse model of vaginal colonization. Female outbred CD1 mice aged 7 to 12 weeks were used forall experiments. Experiments were performed as previously described (5, 8, 9). Briefly, 1 day prior toinoculation (day �1), mice were given an intraperitoneal injection of 0.5 mg �-estradiol valerate (AcrosOrganics) suspended in 100 �l filter-sterilized sesame oil (Sigma) to synchronize estrus. On day 0, micewere vaginally inoculated with 107 CFU of bacteria in 10 �l PBS. On days 1, 2, 3, and 5, the vaginal lumenwas washed with 50 �l sterile PBS, using a pipette to gently circulate the fluid approximately six times.

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To enumerate the bacteria released from the vaginal lumen, the PBS-based lavage fluid was collectedand placed on ice for no more than 30 min before serial dilutions in PBS were plated onto CHROMagarStrepB plates to obtain CFU counts. For enumeration of A909�pbsP cells with the complementationvector, cells were also plated on THY Spec plates to ensure retention of the complementing plasmid.

RNA collection. For RNA collection from the murine host for RNA-Seq, vaginal lavage fluid from micecolonized with bacteria for 48 h was taken directly from the mice and placed into a tube containing 1ml TRIzol LS reagent (Ambion) and placed on ice. Lavage fluid from a total of 10 mice was placed intothe same tube of TRIzol LS reagent, giving a final volume of 1 ml lavage fluid and 1 ml TRIzol LS reagent.These samples were kept on ice for no more than 30 min. Samples were centrifuged for 1 min at14,000 � g, the TRIzol LS Reagent was removed, and RNA preparation was completed using the AmbionRiboPure Bacteria kit (AM1925; Thermo Fisher). Briefly, 250 �l of RNAWiz was used to resuspend thebacterial pellets. RNA was then purified according to the manufacturer’s protocol using 10 min of beadbeating using a MiniBeadbeater (BioSpec) set to homogenize to lyse the cells. Following collection, RNAwas treated with DNase I for 30 min at 37°C. Vaginal lavage fluid from 10 mice generally gave an averageof �0.5 to 5 �g of total RNA. For collection of RNA from planktonic cultures, bacteria were grownstatically overnight at 37°C in THY broth supplemented with the appropriate antibiotics. Followingovernight growth, cultures were back diluted 1:50 in freshly prepared CDM broth. Once the cells reachedan optical density at 600 nm (OD600) value of 0.3 to 0.6, cultures were spun down at the same OD, andRNA was prepared from bacterial pellets as described above using the RiboPure Bacteria kit and treatedwith DNase I. For quantitative PCR (qPCR), planktonic cultures were grown as above, back diluted in CDM,and grown to an OD600 value of 0.3 to 0.6. One milliliter of cells at an OD600 of 0.4 was then pelleted andresuspended in either 1 ml of PBS or 1 ml of vaginal lavage fluid. Cells were incubated at 37°C for 1 hand then pelleted, and RNA was collected and treated with DNase I as described above.

Preparation of eukaryotic and ribosomally depleted cDNA libraries for RNA-Seq. Prior to thelibrary creation, RNA samples collected from the murine vaginal tract were depleted of eukaryotic RNAusing the MICROBEnrich kit (AM1901; Thermo Fisher). Both vaginal and cultured samples were then takenthrough the rRNA removal MICROBExpress kit (AM1905; Thermo Fisher) according to the manufacturer’sinstructions. RNA integrity and eukaryotic and rRNA depletion were then assessed by measurement onboth a TapeStation 2200 (Agilent) and a Qubit RNA high-sensitivity fluorometer (MBL) by the DNAServices Facility (DNAS) at the University of Illinois at Chicago Center for Genomic Research. If a highconcentration of rRNA was still detected, samples were taken through the MICROBExpress kit anadditional time.

RNA-Seq libraries were generated from a small amount of RNA (10 to 400 ng) using the KAPAStranded RNA-Seq Library Preparation kit for Illumina Platforms (KR0934; KAPABiosystems). Briefly, 10 to400 ng of rRNA-depleted RNA was fragmented by heating fragmentation buffer at 94°C for 6 min.First-strand cDNA was synthesized using random primers followed by 2nd-strand synthesis and marking.A poly(A) tail was then added to double-stranded cDNA (dscDNA) fragments, and Illumina adapters wereligated onto the library fragments. The library was then subjected to two rounds of cleanup using

FIG 6 Model of S. agalactiae murine vaginal colonization, illustrating vaginal colonization of GBS,signaling via SaeRS, and host colonization via PbsP.

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magnetic beads for DNA purification (P920-30; 101Bio) followed by library amplification for 10 cycles andan additional magnetic-bead cleanup step. All libraries were then sent to the DNAS facility for qualitycontrol (QC) and quantification on the Tapestation 2200 instrument (Agilent). At least 20 �l of 50 nMlibraries was sent to the University of Chicago Genomics Facility for sequencing and analysis.

RNA sequencing and analysis. Raw sequencing data were analyzed by the University of Illinois atChicago Research Resources Center. Data were aligned to the GBS A909 genome using BWA MEM (40),and gene expression was quantified using featureCounts (41). Gene expression was normalized to countsper million sequences, and differential expression analysis was performed on raw counts using edgeR(42), and the false-discovery rate (FDR) correction was used to adjust P values for multiple testing.Significantly differentially expressed genes (DEGs) with FDR of �0.05 were selected for pathway analysis.The KEGG gene and pathway annotations for A909 (T number T00278) were downloaded from the KEGGwebsite (http://www.genome.jp/dbget-bin/www_bget?gn:T00278), and all genes were assigned to theirKEGG Orthology (KO) group. All metabolic pathways associated with any KO in the A909 genome weredownloaded, and a list of all KOs in each pathway was created to serve as a pathway database.Enrichment statistics for each pathway were computed with respect to our differentially expressed genesby comparing the fraction of KOs in a pathway that were DEGs to the overall fraction of DEGs. Wecomputed P values using Fisher’s exact test and corrected for multiple testing with the FDR correctionover all pathways.

Construction of saeR and pbsP mutants and pLC010 complementation plasmid. For generationof a plasmid to replace saeR with aphA3 (kanamycin resistance gene), upstream and downstream DNAfragments flanking the saeR gene were amplified by PCR using primer pairs LC112/LC115 and LC113/LC116, respectively. These products were purified and then fused in a second PCR using the outsideprimers LC112/LC116. This fusion product was cloned into a temperature-sensitive pJC159 (Cmr) vectorusing internal enzymes HindIII and ClaI following by ligation. Using inverse PCR, this plasmid wasreplicated linearly using primers LC129/LC130 with MluI cut sites between the upstream and down-stream saeR sequences. Separately, the kanamycin resistance gene aphA3 was amplified from pOSKARusing primers JC292/JC304 with MluI cut sites. The linearized plasmid and aphA3 gene were digestedwith MluI and ligated. This plasmid was electroporated into competent BH10C E. coli cells and platedonto LB plates supplemented with Cm and Kan for propagation. The plasmid was electroporated intoelectrocompetent A909 cells, and a two-step temperature-dependent selection process was used toisolate mutants of interest (43). Briefly, cells containing each deletion construct were grown at thepermissive temperature (30°C) and then shifted to 37°C and plated on the THY plates containing Cm andKan to select for bacteria in which the plasmid had integrated at one of the flanking regions. Cells werethen grown at the permissive temperature to allow the plasmid to recombine out of the chromosome,and loss of Cm resistance but maintenance of Kan resistance was used to identify a successful secondcrossover event and loss of the mutation vector. Genotypes were confirmed by PCR and sequencing. Thecreation of A909Δ pbsP::cat was done using Gibson assembly of four fragments. The temperature-sensitive pJC162 (Ermr) vector was digested with EcoRI and PstI. PCR was done on the 984 bpimmediately upstream of pbsP using primers LC169/LC170, on the chloramphenicol resistance gene catusing primers LC171/LC172, and on the 1,070 bp immediately downstream of pbsP using primersLC173/LC174. The three PCR products and the digested vector were combined with 2� HiFi DNAAssembly master mix (E2621; New England BioLabs [NEB]) and incubated at 50°C for 60 min. This mixturewas then electroporated into E. coli BH10C cells and plated onto LB plates supplemented with Erm andCm, and the knockout was created as described above, selecting for Cm colonies that lost the Ermresistance marker.

To create the complementation plasmid pLC010, the pbsP gene was amplified from A909 genomicDNA using primers LC253/LC254. Plasmid pJC303, a pLZ-12-Spec-based plasmid with a constitutive recApromoter, was digested with NotI and BamHI. The digested plasmid and pbsP PCR product wereassembled using a Gibson reaction with 2� NEBuilder Hifi DNA Assembly master mix (E2621; NewEngland BioLabs). The resulting plasmid, pLC010, was electroporated into E. coli BH10C cells. Followingpropagation in E. coli, the plasmid was sequenced and electroporated into electrocompetent A909ΔpbsPcells and recovered on THY Cm, Spec plates.

Preparation of cDNA and qPCR. All primers used in qPCR are listed in Table S2. cDNA for qPCR wasmade using DNase I-treated RNA from cells incubated with either PBS or pooled vaginal lavage fluids.cDNA preparation was done using the Superscript III first-strand synthesis system (18080051; ThermoFisher Scientific) according to the manufacturer’s instructions, including treatment with RNase H.Amplification was done using antisense gene-specific primers LC060 (gyrA), LC133 (pbsP), and LC199(sak_1753). cDNA was diluted between 1:2 and 1:5, depending on the desired concentration, and usedfor qPCR. qRT-PCR was done using the Fast SYBR green master mix (4385612; Applied Biosystems) anda CFX Connect Real Time PCR detection system (1855200; Bio-Rad). The gyrase A gene (gyrA), ahousekeeping gene not seen to have differential expression during growth in the vaginal tract, was usedas a reference gene. All samples were run in triplicate technical replicates on a single plate, and triplicatebiological replicates were used to determine final statistics.

SaeR D53E protein expression and purification. Cloning of saeR D53E into the pET21a expressionvector containing a 6His tag was done using the NEBuilder HiFi DNA assembly master mix (New EnglandBioLabs). A 692-bp gblock fragment of saeR (sak_0467) containing a D53E mutation was ordered fromIntegrated DNA Technologies. The gblock fragment was amplified by PCR using primers LC190/LC191.This product and pET21a vector were digested with XhoI and XbaI. The plasmid and DNA were ligatedusing 2� Hifi master mix (NEB). The resulting plasmid was electroporated into E. coli BL21(DE3) cells andselected on LB agar plates containing ampicillin. BL21 cells containing the expression plasmid were

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grown overnight in LB containing Amp and back diluted to 1:20 into 1 liter fresh LB medium and grownat 37°C. When cells reached an OD600 of approximately 0.5, 1 mM isopropyl-�-D-thiogalactopyranoside(IPTG) was added. Cells were incubated at 28°C for 6 h and then harvested and frozen overnight at�20°C. Cells were lysed in 20 mM Tris (pH 8.0)–100 �g/ml lysozyme using sonication. Lysed cells werespun down, and supernatant was passed through a 0.4-�m filter. Protein was purified using a 3-ml HisPurNi-nitrilotriacetic acid (Ni-NTA) column (88226; Thermo Fisher). Glycerol (20%) was added to purifiedprotein preparations prior to storage at �80°C.

Electrophoretic mobility shift assay (EMSA). Primers used for EMSA probe DNA are listed in TableS2. The pbsP promoter DNA probe (195 bp) was amplified using primers LC203/LC204. LC203 includesa 5= fluorescent 6-carboxyfluorescein (6-FAM) tag (Sigma). Unlabeled pbsP promoter DNA probe (191 bp)was amplified using unlabeled primers LC204/LC142. Unlabeled nonbinding DNA (100 bp) was amplifiedusing unlabeled primers LC231/232. EMSA reaction volumes included 5 nM labeled probe, 50 mM Tris(pH 8.0), 50 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 0.2 mM dithiothreitol (DTT), 0.05% Triton X-100, 12%glycerol, 10 �g bovine serum albumin (BSA), and 50 ng salmon sperm DNA. Protein was added in theconcentrations shown in Fig. 5C. When needed, unlabeled pbsP promoter DNA or unlabeled nonbindingDNA was added as shown in Fig. 5C. Reaction mixtures were incubated at room temperature for 30 min.Xylene cyanol (5%, 1 �l) was added to each reaction mixture, and 10 �l was run on a 6% nondenaturingTris-borate polyacrylamide gel (120-V prerun for 30 min, 120-V run for 75 min). The gel was sandwichedbetween clear polypropylene sheet protectors and imaged using a Typhoon Trio Variable Mode Imager(GE Healthcare) using fluorescent excitation/emission wavelengths of 488/520 nm.

Ethics statement. All mouse experimentation was approved by the University of Illinois at ChicagoAnimal Care and Use Committee (ACC) and IACUC under protocol number 16-068. All animal work wascarried out using accepted veterinary standards in accordance with the Animal Care Policies of theUniversity of Illinois at Chicago Office of Animal Care and Institutional Biosafety Committee and IACUC.This institution has Animal Welfare Assurance Number A3460.01 on file with the Office of LaboratoryAnimal Welfare, NIH.

Accession number(s). Sequence data have been deposited in the NCBI GEO database underaccession no. GSE109680.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00762-17.

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

ACKNOWLEDGMENTSWe acknowledge the University of Chicago Genomics Facility for running the

RNA-Seq samples, Kelly Doran and Katy Patras for instructions on the GBS vaginalanimal model, and Ted Bae for helpful insights into the SaeRS system.

Michael J. Federle is the principal investigator (PI) on NIH grant AI091779 and theBurroughs Wellcome Fund Investigators in Pathogenesis of Infectious Diseases. Laura C. C.Cook was supported by NIH F32AI110047-01. Portions of this project were supported by aChicago Biomedical Consortium Postdoctoral Research Grant. Mark Maienschein-Cline andHong Hu are supported in part by NIH CTSA UL1TR002003. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript.

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