Expression of the Group A Streptococcus Fibrinogen-Binding Protein Mrp Is Negatively Regulated by the Small Regulatory RNA FasX

ABSTRACT

The group A Streptococcus (GAS; Streptococcus pyogenes) causes an elaborate array of human diseases. In part, such variability in disease potential is a consequence of GAS manipulating the expression of a catalogue of virulence factors, with regulation occurring at both the transcriptional and posttranscriptional levels. The GAS small regulatory RNA (sRNA) FasX contributes to this regulatory activity, enhancing expression of the thrombolytic agent streptokinase, and reducing expression of collagen (pili) and fibronectin (PrtF1 and PrtF2) -binding adhesins. Here, we expand insight into the regulatory targets of FasX by identifying the M-related protein (Mrp), a fibrinogen-binding adhesin with anti-phagocytic activity, as a negatively-regulated target of FasX. Importantly, investigation of the consequences of FasX-mediated regulation led to the discovery that FasX is a major positive regulator of GAS survival and proliferation in non-immune whole human blood, with a 30-fold difference in GAS cell numbers between a fasX mutant strain and isogenic parental and complemented mutant strains. No difference in cell numbers were observed when these strains were grown in human serum, consistent with the protective phenotype associated with FasX occurring due to the inhibition of cell (e.g., neutrophil) – mediated GAS killing. The FasX-regulated factor/s responsible for the blood survival phenotype remain to be defined. In summary, we expand the known FasX regulon and identify a new phenotype associated with the regulatory activity of this key GAS sRNA.

IMPORTANCE Small regulatory RNAs (sRNAs) represent a major class of regulatory molecule that promotes the ability of the group A Streptococcus (GAS) and other pathogens to regulate virulence factor expression. Despite FasX being the best-described sRNA in GAS, there remains much to be learned. Here, we highlight the importance of FasX, identifying for the first time that the loss of this sRNA results in a major reduction in the ability of GAS to survive in human blood, a phenotype critical to the ability of this human-specific pathogen to cause severe invasive infections. We also identified a novel regulatory target of FasX, thereby expanding the known regulon of this key sRNA.

INTRODUCTION

The ability to circumvent the host immune response is a critical attribute of bacterial pathogens. From the production of peptidases and proteases that cleave immune system peptides and proteins (1), to the production of adhesins that coat the bacterial cell with host proteins as a means to inhibit complement deposition (2), pathogens express virulence factors that assist in immune evasion. In most cases, these virulence factors are not expressed continuously; rather their expression is highly regulated, with different virulence factors being expressed at specific points during the infection process. Transcriptional and posttranscriptional regulatory systems that control virulence factor expression have thus been identified as key contributors to bacterial pathogenesis (3–5).

The group A Streptococcus (GAS; Streptococcus pyogenes) is a human-specific Gram-positive bacterial pathogen (6). GAS cause a remarkably diverse array of disease manifestations, ranging from mild and self-limiting infections such as pharyngitis and impetigo, to severe infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (7, 8). GAS express a catalogue of immunomodulatory virulence factors with distinct functions. For instance, the C5a peptidase (ScpA) and chemokine protease (SpyCEP) cleave and inactivate the complement proteins C5a/C3/C3a and an array of chemokines, respectively (9–13). The classical GAS virulence factor is the cell wall-anchored M protein, a protein that is highly variable in sequence at its N terminus, with this variation serving as the basis for typing GAS isolates into more than 200 serotypes (M-types) (14, 15). The M protein has anti-phagocytic properties, attributed to its ability to bind inhibitory regulators of the complement system, such as factor H (16). Some M protein types also bind host fibrinogen: coating the GAS cell surface with this protein leads to the inhibition of C3b deposition and hence to the inhibition of complement activation (17). An additional GAS virulence factor is the M-related protein (Mrp), which is structurally similar to the M protein and also binds fibrinogen to the GAS cell surface to inhibit C3b deposition (18, 19).

GAS virulence factor expression is tightly regulated by the activity of up to 14 two-component regulatory systems, more than 40 “stand-alone” transcriptional regulators, and a small number of small regulatory RNAs (sRNAs) (20–24). The best described GAS sRNA is the 205-nt FasX, which is the effector molecule of the FasBCAX regulatory system (25). FasX functions through at least two distinct mechanisms to regulate virulence factor expression. FasX enhances expression of the thrombolytic agent streptokinase (SKA) by increasing the stability of ska mRNA, a process that requires formation of an sRNA:mRNA heteroduplex between FasX and the extreme 5′ end of ska mRNA (26). FasX also serves as a negative regulator, reducing expression of the fibronectin-binding adhesins PrtF1 and PrtF2, and of the collagen-binding GAS pilus (27–29). Negative regulation by FasX occurs following binding of FasX to the prtF1, prtF2, or pilus mRNAs at sites that overlap the ribosome-binding site, with this interaction occluding the ability of ribosomes to bind to the mRNA, reducing mRNA translation.

Here, we present data showing that FasX negatively regulates the expression of Mrp on the GAS cell surface. While the mechanism of regulation remains under investigation, we have identified that the regulation occurs at least in part at the RNA level. In addition, by incubating parental, fasX mutant, and complemented mutant strains in human blood and serum we discovered that FasX protects GAS against cell-mediated killing. Thus, we expand our knowledge of the FasX regulon and identify a new phenotype associated with the regulatory activity of this key sRNA.

RESULTS

In search for a fibrinogen-binding protein whose expression is FasX regulated.

Previously, we identified the mechanisms by which FasX regulates the expression of the virulence factors streptokinase, PrtF1, PrtF2, and pilus (26, 28, 29). PrtF1 and PrtF2 are fibronectin-binding adhesins, while the pilus is a collagen-binding adhesin. The initial study into FasX activity identified that a fasX mutant GAS strain bound fibronectin, collagen, and fibrinogen at a greater level than the parental strain (25). However, our previous transcriptome analysis of the FasX regulon in serotype M1 GAS failed to identify any regulated gene encoding a fibrinogen-binding protein (29). As the initial FasX study was performed using a serotype M49 GAS strain (25), not an M1 strain, we hypothesized that M1 GAS lack the gene/s encoding the fibrinogen-binding protein/s regulated by FasX in M49 GAS. A comparison between the genome sequences of the M1 strain MGAS5005 and the M49 strain NZ131 identified the mrp gene as being present in M49 GAS, absent in M1 GAS, and encoding a fibrinogen-binding protein. The mrp gene is variably present along serotype-specific lines (Fig. 1) and is present in the M28 strain MGAS6180.

FIG 1.

Schematic of the genes located between mga and scpA in representative serotype M1, M2, M6, M28, and M49 GAS strains. A subset of GAS serotypes contains mrp (black) upstream of emm (red).

FasX reduces mrp mRNA levels in the M28 GAS isolate MGAS6180.

To assess whether FasX regulates mrp mRNA abundance we performed quantitative RT-PCR analysis of the parental serotype M28 strain MGAS6180 containing an empty vector, an isogenic fasX mutant derivative containing the empty vector (6180ΔfasX + vector), and a plasmid-complemented mutant derivative (6180ΔfasX + pFasX). The fasX mutant strain produced 1.5-fold greater levels of mrp mRNA, relative to the parental strain; the complemented strain, which overexpresses FasX, produced 5-fold lower mrp mRNA levels relative to the parental strain (Fig. 2A). This regulatory pattern is consistent with that of the FasX-mediated regulation of prtF1 and prtF2 mRNAs (28). In addition to FasX negatively regulating mrp mRNA levels in MGAS6180, it also positively regulates ska mRNA levels, consistent with our previous data (Fig. 2A) (27).

FIG 2.

The FasX sRNA negatively regulates mrp mRNA abundance in the serotype M28 GAS isolate MGAS6180. (A) Quantitative RT-PCR was performed using RNA isolated from triplicate exponential-phase cultures of each strain. Shown is the average (± standard deviation) from three independent experiments. Statistical significance was tested via the Wilcoxon Signed Rank Test, *, P < 0.05; **, P < 0.01. (B) Northern blot analysis of mrp transcripts. Total RNA was isolated from the indicated strains, separated by electrophoresis, transferred to membrane, and probed with an mrp-specific probe. Prior to membrane transfer the RNA was stained in the gel to allow visualization of the 23s and 16s rRNA bands for use as a loading control.

Confirmation that FasX reduces the abundance of mrp mRNA was attained by performing Northern blot analysis. As a negative control for this analysis, we created strain 6180Δmrp (Fig. S1), a derivative of MGAS6180 in which the mrp gene had been deleted. The Northern blot data were similar to that gained by quantitative RT-PCR, with the fasX mutant strain showing a small but consistent increase in mrp mRNA abundance and the FasX-overexpressing strain producing mrp mRNA in lower abundance (Fig. 2B).

mrp is transcribed as a mono-cistronic mRNA from its own promoter and as a poly-cistronic mRNA from a promoter upstream of the mga gene.

The Northern blot data of Fig. 2B identified the presence of at least three different-sized mrp-containing mRNAs (approximately 1.1 kb, 1.4 kb, and 3 kb in length). To begin to investigate the dynamics of mrp transcription, we used 5′ RACE analysis to identify the transcriptional start site and putative promoter region for at least one of the three different mrp mRNAs. The identified transcriptional start site was located 62 nucleotides upstream of the mrp start codon (Fig. 3A). Based upon the location of the promoter and the size of the mrp gene we propose that the 1.4 kb mrp mRNA originates from this promoter. The nature of the 3 kb mrp mRNA was investigated by RT-PCR analysis, testing the hypothesis that it is a consequence of transcriptional read-through from the upstream multiple gene regulator of GAS (mga) gene (Fig. 3B) (30). Using a single reverse primer (R) embedded in the 5′ end of the mrp gene and four different forward primers (F1-F4) located at increasing distances from mrp (Fig. 3B), we generated PCR products for all four reactions (Fig. 3C). Given that primer F4 is located within the mga gene, these data shows that at least some mrp-containing mRNA transcripts also contain mga. The regulatory consequences, if any, to Mrp expression of this observed cotranscription between mga and mrp remains to be investigated, as does the nature of the 1.1 kb mrp-containing mRNA. Possible scenarios for the nature of the 1.1 kb transcript include that it originates at the same site as the 1. 4 kb transcript but terminates at an alternative site within mrp, or that it originates at an uncharacterized promoter within mrp and terminates at the same site as the 1.4 kb transcript.

FIG 3.

The mrp gene is transcribed individually as well as in combination with the upstream gene mga. (A) Identification of the mrp transcriptional start site via 5′-RACE analysis. The bent arrow represents the identified transcriptional start site. The putative -35 and -10 promoter sequences are underlined. The mrp ATG start codon is boxed. The putative ribosome-binding site is shown in italics. (B) Schematic of the mga and mrp genes with the locations of the primers used in RT-PCR analysis being highlighted with arrows (primers R and F1 through F4). (C) RT-PCR analysis of mrp transcription. The four different templates (water [negative control], genomic DNA [gDNA; positive control], cDNA-RT [to control against gDNA contamination of the RNA], and cDNA+RT [test]) were each used in conjunction with four primers pairs (primer R with primers F1 through F4) in PCR. The PCRs were separated on 2% agarose gels and visualized.

FasX reduces the abundance of Mrp on the GAS cell surface.

In order to explore whether the regulation of mrp mRNA abundance by FasX is also observed at the protein level, we performed Western blot analysis using an anti-Mrp antibody. Cell wall protein fractions of the parental, mutant, and complemented mutant strains, as well as of the isogenic mutant strain 6180Δmrp, were isolated for analysis. The Western data mirrors the results at the mRNA level: namely, that an increase in Mrp levels is observed following fasX mutation (Fig. 4A), and that increased FasX expression via pFasX leads to a reduction in Mrp levels.

FIG 4.

FasX negatively regulates Mrp expression on the GAS cell surface. Western blot analysis using an anti-Mrp antibody in conjunction with cell wall protein fractions. (A) samples gained from the parental M28 isolate MGAS6180 (MGAS6180 + vector), fasX mutant derivative (6180ΔfasX + vector), complemented mutant derivative (6180ΔfasX + pFasX), and mrp deletion mutant derivative (6180Δmrp + vector). (B) samples gained from the parental M28 clinical isolate MGAS6180 (MGAS6180 + vector), mrp deletion mutant derivative (6180Δmrp + vector), parental M2 isolate MGAS10270, fasX mutant derivative (10270ΔfasX + vector), and complemented mutant derivative (10270ΔfasX + pFasX). As loading controls, the membranes used in Western blot analyses were stained, with representative subsets of the proteins shown. All samples were isolated at least in triplicate, and all samples were used at least twice in Western blot analysis, with representative data shown.

To test whether the FasX-mediated regulation of Mrp production is restricted to serotype M28 isolates, or is observed more broadly in the GAS population, we repeated the Western blot analysis but this time isolated cell wall protein samples from an mrp-containing serotype M2 isolate, a fasX mutant derivative, and a complemented mutant derivative. While the level of Mrp made in the M2 background strains were overall lower than observed in the M28 background, the pattern of Mrp regulation by FasX remained the same, with the fasX mutant strain showing higher Mrp levels than the parental strain, and the higher FasX levels due to plasmid pFasX resulting in lower Mrp production (Fig. 4B). Thus, our data are consistent with the FasX-mediated negative regulation of Mrp abundance being a general feature in GAS isolates that harbor the mrp gene and a functional Fas system.

FasX reduces fibrinogen-binding by GAS in a manner similar to the regulation of Mrp production.

Given that FasX inhibits cell surface Mrp production, and that Mrp is a fibrinogen-binding protein, we next tested whether FasX inhibits the ability of GAS to bind fibrinogen. Fibrinogen-coated ELISA plate wells were used to assess the ability of our parental M28 strain and fasX mutant, fasX complemented mutant, and mrp mutant derivatives to bind to the immobilized fibrinogen. GAS adherence followed a pattern that mirrored Mrp abundance, with the fasX mutant strain adhering at a higher level than the parental strain, and the fasX complemented mutant strain adhering at a lower level (Fig. 5). Also of note, similar levels of binding were gained for the complemented fasX mutant strain and the mrp deletion mutant strain. While not proven, as mrp and fasX double mutant strains were not used, the data are consistent with FasX inhibiting the ability of GAS to bind fibrinogen due to the FasX-mediated inhibition of Mrp production.

FIG 5.

FasX inhibits GAS adherence to fibrinogen. Diluted cultures of the indicated GAS strains were added to fibrinogen-coated wells of an ELISA plate and the percentage of CFU that adhered were calculated. Shown are the mean values (± standard error of the mean) for each strain gained from seven biological replicates. ANOVA was performed with pairwise strain comparisons investigated using the Tukey multiple-comparison test, with the P values shown. N.S., not significant.

FasX enhances the ability of GAS to survive and proliferate in human blood.

Given that Mrp inhibits the neutrophil-mediated killing of GAS (17, 31), we set out to test the hypothesis that FasX, which inhibits Mrp production, reduces the ability of GAS to survive in human blood. Through use of Lancefield bactericidal assays, we identified that a 30-fold lower multiplication factor was observed for the fasX mutant strain relative to the parental and complemented strains (Fig. 6). Thus, FasX is a major positive regulator of GAS survival in blood, a finding that was the opposite of our hypothesis (see discussion section). To assess whether the protective effect of FasX is due to the inhibition of cell- or serum-mediated GAS killing we repeated the assays using human serum. All three tested GAS strains were essentially identical in their survival pattern in human serum (Fig. 6), consistent with the mechanism of FasX-mediated protection involving the inhibition of GAS killing by phagocytic cells.

FIG 6.

FasX protects against the cell-mediated killing of GAS in human blood. The parental, mutant, and complemented mutant strains were compared in Lancefield bactericidal assays. The survival of each strain after 3 h growth in aliquots of heparinized non-immune human blood or non-immune human serum was determined using the calculation (number of CFU after 3 h/number of CFU at 0 h). The experiment was performed in triplicate, with mean values (± standard deviations) shown. Statistical significance was determined via ANOVA (overall ANOVA: P = 0.0009 for blood data, P = 0.4326 for serum data). Individual strains were compared as indicated using the Tukey’s multiple-comparison test. **, P < 0.01; n.s., not significant.

DISCUSSION

Precise regulation of virulence factor expression is key to the ability of bacterial pathogens to circumvent the host immune response and cause disease. Here, we show that GAS use a small regulatory RNA, FasX, to fine-tune expression of the fibrinogen-binding protein Mrp. Together with the FasX-mediated repression of the collagen-binding pilus (29), and of the fibronectin-binding proteins PrtF1 and Prtf2 (28), this highlights FasX as a major negative regulator of adhesin expression in GAS. Given that FasX also positively regulates expression of SKA, which indirectly degrades blood clots and tissue barriers, the available data are consistent with FasX functioning at the interphase between colonization and dissemination (Fig. 7).

FIG 7.

Graphical representation of the Fas regulatory system. Solid lines represent known regulatory activity, while dashed lines represent hypothesized regulatory activity. The four genes of the fas locus, fasBCAX, are represented by block arrows. The FasBCA proteins form an as-yet-uncharacterized regulatory system that is required for significant FasX production (25) (Sumby, unpublished data).

The mrp gene is variably present among GAS isolates, with its presence and absence being distributed along serotype-specific lines (e.g., Fig. 1). Mrp is a fibrinogen-binding protein that coats the GAS cell surface with fibrinogen, resulting in the inhibition of C3b deposition and therefore of complement activation (18, 32). Given this activity, and our finding that FasX negatively regulates Mrp expression (Fig. 4) and fibrinogen-binding activity (Fig. 5), this led us to hypothesize that FasX reduces the ability of GAS to avoid cell-mediated killing in human blood. However, we discovered a 30-fold increase in GAS cell abundance after 3 h of incubation in blood for the parental and complemented strains relative to the fasX mutant strain (Fig. 6). Thus, despite the finding that FasX reduces the ability of GAS to coat itself with fibrinogen (Fig. 5), FasX promotes, rather than inhibits, GAS survival and proliferation in blood. No appreciable difference between the tested strains in human serum is consistent with FasX promoting resistance to cell (e.g., neutrophil) -mediated killing in blood (Fig. 6), rather than resistance to the antimicrobial factors present in human serum.

The disconnect between FasX inhibiting Mrp expression but enhancing GAS survival in human blood may be explained by the activity of other FasX-regulated virulence factors. For example, the inhibition of pilus expression by FasX could account for the observed phenotype as, at least in serotype M1 and M3 strains, pili have been identified as inducers of cell-mediated GAS killing (33–36). The molecular basis of the protective effect of FasX (i.e., which FasX-regulated virulence factor/s are responsible for the observed phenotype) is under investigation. In this regard, we have preliminary proteomic data (unpublished) congruous with FasX not only negatively regulating expression of pili, PrtF1, PrtF2, and Mrp but also the integrin-binding protein R28 (37), the collagen-binding protein Scl1 (38), and the fibronectin-binding protein SfbX (39), consistent with the FasX regulon being larger than currently appreciated. Validation of our proteomic data are ongoing, and may assist in our efforts to characterize the molecular basis for the FasX-mediated blood survival phenotype.

While the mechanism of Mrp regulation by FasX was not determined in this study, we did identify that the regulation was seen at both the RNA and protein levels (Fig. 2 and 4). One possibility is that FasX inhibits mrp mRNA translation, similar as occurs in the regulation of PrtF1, PrtF2, and pilus expression (27–29). Such a mechanism of regulation may also be observed at the RNA level as a reduction in the number of ribosomes on mrp mRNA could lead to a decrease in the stability of the mRNA, as ribosomes are known to have a protective effect on mRNA stability (40). However, rifampicin RNA stability assays, similar to those we performed previously (26), did not uncover any difference in stability between the presence and absence of FasX (data not shown). An alternative possibility for the mechanism of FasX-mediated regulation is that FasX alters the processing of mrp mRNA. However, the Northern blot data of Fig. 2B shows similar-sized mrp transcripts regardless of the presence or absence of FasX, which reduces the likelihood that alternative processing is the mechanism of action. Thus, the mechanism of regulation of Mrp abundance by FasX remains to be elucidated but appears to be a mechanism that has not previously been described for FasX. Given that Mrp is being investigated as a possible GAS vaccine antigen (41), insight into the mechanism by which FasX regulates cell surface Mrp expression may inform with regard to the suitability of targeting this protein.

We identified that some mrp transcripts arise via transcriptional read-through from the upstream mga gene (Fig. 3). Mga is a stand-alone transcription factor that positively regulates transcription of key GAS virulence factor-encoding genes, including each of the virulence factor-encoding genes shown in Fig. 1 (42). The regulatory consequences, if any, of mga-mrp polycistronic transcripts is unclear. Interestingly, while mga-mrp polycistronic transcripts have not been previously described, mga-emm polycistronic transcripts have been (43). In work performed in a serotype M12 strain background, which lacks the mrp gene and has a similar gene content between mga and scpA as M1 GAS (Fig. 1), Bormann and Cleary identified that transcriptional read-through from mga into emm occurred at a high rate (43). Thus, read-through appears to be a common phenomenon for mga and the gene downstream.

In summation, we have expanded the known regulon of the virulence-regulating GAS small regulatory RNA FasX, adding inhibition of Mrp expression to its regulatory activity. Furthermore, we discovered that the regulation afforded by FasX enhances the ability of GAS to survive and proliferate in human blood, an attribute that positively correlates with invasive disease severity. Given the magnitude of the FasX-mediated blood survival phenotype (30-fold; Fig. 6), the future delineation of its molecular basis will be of value to both basic and clinical researchers.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

The GAS strains used in this study are listed in Table S1. Routine growth of liquid GAS cultures made use of Todd-Hewitt broth with 0.2% yeast extract (THY broth), and cultures were incubated at 37°C (5% CO₂). Chloramphenicol (4 μg/mL) and/or spectinomycin (150 μg/mL) were added when required.

Quantitative RT-PCR analyses.

Total GAS RNA was isolated from the indicated GAS strains, as previously described (44). Briefly, triplicate cultures of each GAS strain were grown to the exponential phase of growth (corresponding to an optical density at 600 nm [OD600] of 0.5) in THY broth. One volume of GAS culture was subsequently added to 2 volumes of RNAprotect Bacteria reagent (Qiagen Inc.) and incubated at room temperature for 5 min. Following centrifugation at 5,000 × g for 10 min at 4°C, the bacterial pellets were snap-frozen in liquid nitrogen and stored at −80°C until they were ready for use. Bacterial cells were processed for RNA isolation using a mechanical lysis method in conjunction with an RNeasy minikit (Qiagen Inc.). The quality and quantity of the isolated RNA were assessed by using a Bioanalyzer 2100 system (Agilent Technologies). cDNA was synthesized from total GAS RNA using the Superscript III (ThermoFisher) reverse transcriptase as per the manufactures’ instructions. TaqMan quantitative RT-PCR was performed using a CFX Connect real-time system (Bio-Rad). Gene transcript levels were compared between strains using the ΔΔCT method. TaqMan primers and probes for the genes of interest, and the internal control gene tufA, are listed in Table S2.

Creation of the mutant GAS strain 6180Δmrp.

To facilitate investigation of whether FasX regulates expression of Mrp we created an mrp deletion mutant derivative of our parental GAS isolate MGAS6180 (Fig. S1) (45). Using the primers listed in Table S2, we generated a plasmid construct that was used to replace the mrp gene in MGAS6180 with a nonpolar spectinomycin resistance cassette, similar to previously created mutant GAS strains in our laboratory (36, 46). The resultant strain, 6180Δmrp, was verified by PCR, targeted sequencing, and Western blot analyses.

Northern blot analysis of mrp mRNA transcripts.

Total RNA was isolated from exponential-phase GAS cultures as described above. After quantification, 10 μg of each RNA sample was separated on a glyoxal gel using the NorthernMax-Gly kit (ThermoFisher), as per the manufacturers’ instructions. After separation via electrophoresis the RNA was visualized by staining the gel with Radiant Red (Sigma-Aldrich); this was performed so the resultant image could serve as a loading control. Subsequently, the RNA was transferred to Hybond N+ membrane (GE Healthcare) and the membrane probed as described for the NorthernMax-Gly kit (ThermoFisher). The mrp probe used in this study was generated by PCR using the primers listed in Table S2, with biotin-16-dUTP (Sigma-Aldrich) added to the reaction resulting in a biotinylated PCR product. After hybridization in ULTRAhyb buffer (ThermoFisher) and washing with both high and low stringency wash buffers (ThermoFisher), IRDye 680RD streptadavin (1:10,000 dilution; LI-COR) was incubated with the blot to enable detection of the biotinylated probe through use of an Odyssey near-infrared system (LI-COR).

5′ – RACE analysis of the mrp transcriptional start site.

The transcriptional start site (TSS) of mrp was identified by utilizing the 5′ system for rapid amplification of cDNA ends kit (5′-RACE; v2; ThermoFisher) according to the manufacturer's specifications. Briefly, 3 μg of total RNA was used for first-strand cDNA synthesis, generated by reverse transcriptase, using the gene-specific primer (GSP1) for mrp listed in Table S2. The generated cDNA was RNase-treated and column-purified, and the 3′ end was tagged using terminal deoxynucleotidyl transferase and dCTP to create homopolymeric tails of C nucleotides. The product was subsequently amplified using one primer complementary to the dC tail (AAP; kit-supplied) and one primer specific to mrp (GSP2; Table S2); amplicons were gel extracted and cloned into E. coli (TOPO TA; ThermoFisher) for sequencing analysis to identify the transcriptional start site.

RT-PCR analysis of mrp mRNA transcripts.

Total RNA was isolated from MGAS6180 as described above and used to synthesize cDNA using Superscript III (ThermoFisher). An identical reaction but lacking reverse transcriptase (-RT) was also set up to serve as a control against genomic DNA contamination. Four sets of primer pairs (Table S2) were used with the generated cDNA (cDNA+RT), the no RT control (cDNA-RT), genomic DNA (as a positive control), and water (as a negative control). The products of the PCRs were separated on a 2% agarose gel and imaged.

Isolation of cell-wall protein fractions.

Triplicate cultures of each GAS strain (derivatives of MGAS6180 and MGAS10270 (47)) were grown to the mid-exponential phase of growth (corresponding to an O.D.600 of 0.5) in THY broth. Cells were pelleted by centrifugation (4,000 g for 10 min) and washed once with 10 mL TE buffer before resuspending in 1 mL of TE-sucrose buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 20% sucrose, 2 mg/mL lysozyme, and 400U of mutanolysin). Samples were incubated at 37°C with end-to-end rotation in 1.5 mL tubes for 2 h. Samples were centrifuged 15,000 g for 5 min to pellet the protoplasts, and the supernatants were removed to clean 1.5 mL tubes. The centrifugation was repeated to pellet any insoluble material carried over, and the supernatant was analyzed by SDS-PAGE.

Western blot analysis.

Protein samples were separated on a 12% Tris-HCl gel before transferring to nitrocellulose membrane (Transblot semidry system, Bio-Rad). The blot was blocked with 5% milk in PBST (PBS containing 0.1% Tween 20). The primary antibody against Mrp was generated in rabbits using E. coli-purified recombinant protein as the antigen (Pacific Immunology). The polyclonal primary antibody was used at 1:2,000 dilution in PBST for 4 h at room temperature, before washing three times (5 min each) with PBST. The blot was then incubated with anti-rabbit IgG secondary antibody labeled with Alexa Fluor 680 (LI-COR), and an Odyssey system (LI-COR) was used to capture and visualize the fluorescent signal. All Western blots were performed at least twice for all isolated cell-wall protein samples isolated above.

Fibrinogen Binding Assays.

Wells of a 96-well ELISA plate were coated overnight at 4°C using 100 μL of purified fibrinogen (100 ng/μL) or BSA (100 ng/μL). The wells were then washed four times with 1× PBS before blocking with 1% BSA for 1 h at 37°C, and subsequently washed an additional four times with 1× PBS. THY broth cultures of individual GAS strains were grown to the mid-exponential phase of growth (O.D.600 of 0.5) and diluted down to a concentration of ~10⁶ CFU/mL. Wells (both fibrinogen and BSA) then had 100 μL of bacterial suspension added, and the plate was incubated at 37°C for 30 min. After incubation, the wells were washed four times with 1× PBS to remove nonadherent GAS cells. To recover adherent GAS from the wells, each well was treated with 50 μg of trypsin (in 100 μL) and incubated at 37°C for 10 min. The percentage of fibrinogen-binding bacteria was calculated after plating serial dilutions of both the original inoculums and the cells recovered from the wells after trypsinization, on blood agar plates (([no. of CFU recovered from fibrinogen well – no. of CFU recovered from BSA well]/no. of CFU in inoculum) × 100). The experiment was performed a total of seven times (with independently grown cultures each time) and mean data are presented.

Lancefield bactericidal assays.

To test the ability of individual GAS strains to survive in human blood, we performed Lancefield bactericidal assays. Cultures of each strain were grown to early exponential phase (an O.D.600 of between 0.15 and 0.20). Each GAS culture was diluted to 10⁻⁴ in sterile PBS, and 450 μL of whole heparinized blood was added to 50 μL of diluted culture. These mixtures were then incubated for 3 h at 37°C with end-over-end rotation. Fifty μL of each inoculum was simultaneously plated on blood agar plates to allow enumeration the next day. Following incubation, the GAS-blood cultures were diluted and plated on blood agar plates. All samples were incubated overnight at 37°C in a 5% CO₂ atmosphere. The number of CFU was calculated by the formula (number of surviving CFU/initial number of CFU). To assess whether FasX inhibited the cell- or serum-mediated killing of GAS, these assays were also performed using sera, rather than whole blood, from the same donors. The Lancefield assays were performed on three independent occasions using blood from two independent donors (one male and one female) each time, with averaged data shown.