ABSTRACT
Bacterial virulence factor production is a highly coordinated process. The temporal pattern of bacterial gene expression varies in different host anatomic sites to overcome niche-specific challenges. The human pathogen group A streptococcus (GAS) produces a potent secreted protease, SpeB, that is crucial for pathogenesis. Recently, we discovered that a quorum sensing pathway comprised of a leaderless short peptide, SpeB-inducing peptide (SIP), and a cytosolic global regulator, RopB, controls speB expression in concert with bacterial population density. The SIP signaling pathway is active in vivo and contributes significantly to GAS invasive infections. In the current study, we investigated the role of the SIP signaling pathway in GAS-host interactions during oropharyngeal colonization. The SIP signaling pathway is functional during growth ex vivo in human saliva. SIP-mediated speB expression plays a crucial role in GAS colonization of the mouse oropharynx. GAS employs a distinct pattern of SpeB production during growth ex vivo in saliva that includes a transient burst of speB expression during early stages of growth coupled with sustained levels of secreted SpeB protein. SpeB production aids GAS survival by degrading LL37, an abundant human antimicrobial peptide. We found that SIP signaling occurs during growth in human blood ex vivo. Moreover, the SIP signaling pathway is critical for GAS survival in blood. SIP-dependent speB regulation is functional in strains of diverse emm types, indicating that SIP signaling is a conserved virulence regulatory mechanism. Our discoveries have implications for future translational studies.
INTRODUCTION
Bacterial pathogens colonize different host anatomic sites with various ecologies and encounter unique challenges in each tissue microenvironment. The host employs niche-specific anatomic barriers, antimicrobial defense mechanisms, and nutritional immune mechanisms to inhibit bacterial proliferation. Successful pathogens have sensory mechanisms to monitor environmental cues and mount tailored transcriptional responses to adapt to new environments. One such sensory program in Gram-positive bacteria, known as quorum sensing, uses secreted bacterial peptide signals to monitor the population density and mediate the spatiotemporal regulation of virulence factor production (1, 2). The quorum sensing pathway involves signal production, secretion, and sensing by neighboring bacteria and population-wide modulation of gene expression (1–3). Quorum sensing pathways control several bacterial traits, including virulence, biofilm formation, antibiotic resistance, sporulation, and genetic competence (2–7). Virulence regulation by quorum sensing has been studied extensively in vitro. However, bacterial growth in laboratory medium does not fully recapitulate the growth in the complex host environment in which the signaling occurs during infection. Thus, elucidation of the niche-specific contribution of quorum sensing pathways during infection is critical to understand the dynamics of quorum sensing regulatory pathways in vivo. In addition, knowledge of the spatiotemporal pattern of signaling in vivo may lead to interference strategies targeting the quorum sensing pathways for antimicrobial or vaccine development.
Streptococcus pyogenes, also known as group A streptococcus (GAS), is a versatile human pathogen that colonizes diverse host anatomic sites. GAS causes a range of disease manifestations (8, 9), including mild pharyngitis and skin infections and life-threatening invasive infections, such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS also causes acute rheumatic fever (ARF), rheumatic heart disease (RHD), and poststreptococcal glomerulonephritis (10–12). Despite the significant morbidity and mortality associated with GAS infections worldwide (10, 13), disease prevention efforts are significantly hampered by the lack of a licensed human GAS vaccine (14, 15). Thus, continued study of virulence regulatory pathways is warranted to identify additional molecular targets and aid vaccine development.
A secreted cysteine protease known as SpeB is a major virulence factor that is crucial for GAS pathogenesis in multiple anatomic sites (16–20). SpeB is produced abundantly during infection, and its protease activity contributes significantly to host tissue damage and disease dissemination (16, 17, 21, 22). Consistent with this, inactivation of SpeB attenuates virulence in several animal models of infection (18–21, 23, 24). Recently, we discovered that a noncanonical GAS quorum sensing pathway controls speB expression in coordination with bacterial population density (4, 25). A GAS-encoded leaderless peptide signal designated SpeB-inducing peptide (SIP) and an intracellular peptide-sensing global transcription regulator known as regulator of protease B (RopB) form a peptide signal and receptor pair and activate speB expression during high bacterial population density (4). The 8-amino-acid SIP is produced during high bacterial population density, secreted, and reimported into the bacterial cytosol and engages in direct interactions with cytosolic RopB (4) (Fig. 1). SIP binding induces allosteric changes in RopB, resulting in high-affinity interactions with the operator sequences located within the speB promoter and RopB oligomerization (Fig. 1). Subsequently, the transcriptionally competent association between RopB-SIP and the speB promoter induces robust speB expression (3, 4) (Fig. 1). Importantly, the SIP signaling pathway is active during invasive infection in mouse models, and each component of the SIP signaling pathway is critical for GAS pathogenesis (4).
Proposed model for mechanism of SIP-dependent intercellular communication and GAS virulence regulation. At a high cell density, SIP is produced, secreted, and reimported into the cytosol. The high-affinity RopB-DNA interactions and RopB polymerization aided by SIP binding lead to upregulation of SIP gene expression, which results in the robust induction of SIP production by a positive-feedback mechanism. Finally, the SIP-dependent upregulation of speB leads to the abundant secretion of the mature form of SpeB protease (SpeBM), which facilitates host tissue damage and disease dissemination by cleavage of various host and GAS proteins. The ropB and speB genes are divergently transcribed. The block arrows indicate the coding regions of ropB, speB, and the gene for SIP. The angled arrows above the line indicate two transcription start sites for speB, designated P1 and P2. The pseudo inverted repeats containing the RopB-binding site within the speB promoter are marked by arrows and colored in red.
Pharyngitis is the most common form of GAS disease. Oropharyngeal GAS colonization is a major predisposing factor for the development of immunopathological consequences, such as ARF and RHD (12, 26–28). Saliva is the first line of host defense in the oral cavity and contains several innate and adaptive immune factors that control microbial growth (29). However, GAS successfully proliferates and persists in human saliva, and GAS transmission between hosts typically occurs through saliva (30–34). Similarly, development of systemic infection requires that GAS survive in human blood. Previous studies indicated that SpeB is critical for GAS survival ex vivo in human saliva and blood (30). However, the regulatory mechanisms controlling SpeB biogenesis, their contributions to GAS survival ex vivo in human saliva and blood, and their role in oropharyngeal GAS colonization remain unknown. Here we used biochemical analyses, ex vivo gene expression studies, mouse infection studies, and immunologic methods to demonstrate that the SIP signaling pathway is active during GAS growth ex vivo in human saliva and blood and controls speB expression. We discovered that GAS has a distinct speB expression profile during growth ex vivo in human saliva, which may be crucial for pathogen survival in the human host. Importantly, the SIP signaling pathway is functional among strains of diverse GAS emm types and contributes to GAS persistence in human saliva and mouse oropharyngeal GAS colonization. In summary, our findings reveal a ubiquitous role for SIP-mediated SpeB production in GAS pathogenesis in multiple host niches and suggest new therapeutic strategies.
RESULTS
Kinetics of speB transcripts during GAS growth ex vivo in human saliva.To determine the speB expression pattern during GAS growth in vitro, we analyzed GAS growth kinetics and the speB transcript level in GAS grown in Todd-Hewitt broth containing 0.2% (wt/vol) yeast extract (THY). The initial induction of speB expression occurred only during high bacterial population density (6.9 × 108 CFU/ml; 695-fold induction in speB expression), and the speB transcript level persisted at higher levels during stationary phase of GAS growth (Fig. 2A). Next, we assessed growth kinetics and speB transcript levels during GAS growth ex vivo in human saliva. GAS growth increased during the first 16 h and reached a maximum population density (∼107 CFU/ml) at 16 h postinoculation (Fig. 2B). Subsequently, GAS entered into a phase of persistence and sustained viability at a lower population density (∼105 CFU/ml) until 6 days postinoculation (Fig. 2B). Correlation of speB transcript levels with GAS growth in saliva showed a density-dependent pattern of speB expression (Fig. 2B). Compared to the starting time point, the initial induction of speB expression occurred at 12 h postinoculation (2.4 × 106 CFU/ml; 1,169-fold induction of speB expression). speB transcript levels peaked at 16 h postinoculation, corresponding to the highest GAS population density in saliva (1.1 × 107 CFU/ml; 2,533-fold induction of speB expression) (Fig. 2B). speB transcript levels decreased drastically by 48 h postinoculation and remained at basal levels as the number of GAS CFU in saliva declined (Fig. 2B).
GAS growth and speB transcript level kinetics in human saliva. The MGAS10870 strain was grown to mid-exponential phase (A600, ∼0.4), washed twice in sterile PBS, and suspended in human saliva at a starting bacterial population density of ∼105 CFU/ml. Samples were collected at the indicated time points to determine the number of CFU, speB transcript levels, and SpeB protein levels. (A and B) The kinetics of GAS growth and speB transcript levels in THY medium (A) and human saliva (B) are shown. The left y axis represents the growth curve, as determined by assessing the number of CFU by plating serial dilutions of 100-μl aliquots collected at the indicated time points. The right y axis represents the fold change in speB transcript levels at the indicated time points, as measured by qRT-PCR. The fold change in transcript levels relative to the level in the starting culture (time point = 0 h) is shown. (Inset) The exponential phase of GAS growth in saliva is shown. The data graphed are means ± standard deviations for three biological replicates. (C) Western immunoblot analysis of secreted SpeB during growth in human saliva. The cell-free saliva samples were resolved on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were probed with anti-SpeB polyclonal rabbit antibody and visualized with chemiluminescence. The mature form of purified recombinant SpeB (SpeBM; 25 kDa) was used as a marker. The masses of the molecular mass markers are shown in kilodaltons.
We next compared the kinetics of speB transcript levels in GAS grown in saliva and nutrient-rich laboratory (THY) medium. Under both growth conditions, GAS upregulated speB expression in concert with an increase in the bacterial population density (Fig. 2A and B). However, significant differences in the speB expression profiles were observed between the two growth conditions. In saliva, initial induction of speB expression occurred at a much lower population density (2.4 × 106 CFU/ml; 1,169-fold induction in speB expression) relative to GAS growth in laboratory medium (6.9 × 108 CFU/ml; 695-fold induction in speB expression) (Fig. 2A and B). Importantly, even at a much higher bacterial population density (4.3 × 108 CFU/ml; no induction in speB expression), no upregulation of speB expression was observed during in vitro growth (Fig. 2A). Further, in saliva, speB expression increased at between 12 and 24 h postinoculation, and the induction of speB expression was transient (Fig. 2B). In contrast, during GAS growth in laboratory medium, the transcript levels of speB continued to increase along with the increase in bacterial population density.
Finally, to understand the downstream consequences of the transient induction of speB expression on GAS growth in saliva, we assessed secreted SpeB protein levels by Western immunoblotting. The secreted SpeB protease levels resembled the speB expression profile, as SpeB was initially detected at 12 h postinoculation and reached maximal levels at 24 h postinoculation in saliva (Fig. 2C). However, unlike the speB transcript profile, the secreted SpeB protease levels persisted in saliva during the entire 6-day study period (Fig. 2C). Collectively, these data suggest that GAS employs a pattern of speB expression during growth ex vivo in human saliva distinct from the pattern of growth in vitro, and early induction of speB expression and sustained secreted SpeB protease levels may contribute to GAS survival in human saliva.
SIP signaling is active during ex vivo growth in human saliva.To test the hypothesis that the SIP signaling pathway controls speB expression during GAS growth ex vivo in human saliva, we performed synthetic SIP (SIP; MWLLLLFL) addition experiments using the isogenic SIP* mutant strain. In this mutant, the start codon of SIP was replaced with a stop codon, thereby disrupting the translation of SIP (4) (see Fig. S1 in the supplemental material). Thus, in the SIP* mutant strain endogenous SIP production is defective and speB expression is dependent on exogenously added synthetic SIP (4). A scrambled peptide (SCRA; LLFLWLLM) with an amino acid composition identical to that of SIP but in which the order of the sequence was varied was used as a negative control (4). The SIP* mutant strain was grown to late exponential growth phase (A600, ∼1.5) in THY medium, washed with phosphate-buffered saline (PBS), and suspended in an equal volume of fresh human saliva. After 30 min of incubation, cells were supplemented with either synthetic SIP or SCRA and speB transcript levels were measured by quantitative reverse transcription-PCR (qRT-PCR). Consistent with our hypothesis, addition of SIP caused the significant induction of speB expression. In contrast, SCRA failed to induce speB expression (Fig. 3). These results suggest that SIP signaling occurs during GAS growth in human saliva and that SIP is responsible for the upregulation of speB expression in saliva.
SIP signaling occurs during GAS growth in human saliva. Addition of synthetic SIP restores speB expression in the isogenic SIP* mutant strain during growth in human saliva. Scrambled peptide (SCRA), with a length and amino acid composition identical to those of SIP but with the order of the amino acid sequence being varied, was used as a negative control. The SIP* mutant strain was grown to late exponential phase (A600, ∼1.5) in THY, washed in sterile PBS, resuspended in an equal volume of human saliva, and incubated for 30 min. Cells were supplemented with either 500 nM the indicated peptides or the solvent used in peptide stock solutions (DMSO) and incubated for an additional 30 min at 37°C. Transcript levels of speB were assessed by qRT-PCR. The fold change in speB transcript levels relative to the level in unsupplemented GAS growth is shown. The data graphed are means ± standard deviations for three biological replicates.
The SIP signaling pathway promotes GAS growth ex vivo in human saliva.Given that SIP-dependent upregulation of speB expression occurs during GAS growth in saliva, we hypothesized that the SIP signaling pathway is required for optimal growth in saliva. To test this hypothesis, we compared the growth kinetics of the wild-type (WT) parental strain with that of the isogenic ΔspeB, ΔropB, or SIP* mutant strain. All four strains had similar growth kinetics during growth in laboratory medium (4). However, compared to the growth of the WT parental strain, all three mutant strains were significantly impaired in growth in saliva. In addition, the mutant strains persisted at a significantly lower population density (Fig. 4A). Importantly, the defective growth of the isogenic SIP* mutant strain in saliva was restored to the WT-like phenotype in the trans-complemented strain (SIP*::pDC-SIP) (Fig. 4A and S1). Collectively, these data suggest that gene regulation by the SIP signaling pathway contributes significantly to the ability of GAS to persist in saliva.
The SIP signaling pathway aids bacterial survival in human saliva and contributes significantly to GAS colonization of the mouse oropharynx. (A) Growth curves of the indicated strains in human saliva. GAS grown to stationary phase (A600, ∼1.7) in laboratory medium was washed with sterile PBS and resuspended in human saliva. Growth was monitored by plating serial dilutions of 100-μl aliquots at the indicated time points postinoculation. Colonies were counted to determine the number of CFU. The data graphed are means ± standard deviations for three biological replicates. P values (**, P < 0.01) for the indicated strains were determined by comparison to WT GAS. (B) The SIP signaling pathway contributes significantly to GAS colonization of the mouse oropharynx. The percentage of mice with GAS isolated from the oropharynx at the indicated time points is shown. Adult outbred CD-1 mouse (20 mice per group) nostrils were inoculated with 2 × 108 CFU of the indicated GAS strains. The mouse oropharynx was swabbed daily, and the swabs were plated on streptococcal selective agar (SSA). The plates were incubated for 48 h, and beta-hemolytic colonies were counted. P values (****, P < 0.0001) for the indicated strains were determined by comparison to WT GAS.
The SIP signaling pathway contributes significantly to mouse oropharyngeal GAS colonization.We next tested the hypothesis that the SIP signaling pathway contributes to GAS colonization of the mouse oropharynx. Although GAS is a human-only pathogen, the murine model of oropharyngeal GAS colonization has been extensively used to investigate GAS survival at the oropharynx (35–39). Mice were infected intranasally with 108 CFU of each GAS strain. The mouse oropharynx was swabbed daily, and GAS colonization was determined by assessing the number of CFU in throat swabs. Significantly fewer mice were colonized by the isogenic ΔropB, ΔspeB, and SIP* mutant strains than by the WT parental strain (Fig. 4B) (P < 0.0001). However, the defective colonization by the SIP* mutant strain was restored to WT colonization levels in the trans-complemented (SIP*::pDC-SIP) strain (Fig. 4B) (P < 0.0001). Together, these data suggest that the SIP signaling pathway contributes significantly to the ability of GAS to colonize the mouse oropharynx.
SIP signaling occurs during growth ex vivo in human blood, and inactivation of the SIP signaling pathway results in decreased GAS survival in blood.We previously demonstrated that inactivation of SIP attenuated GAS virulence in a mouse model of bacteremia (4). This result led us to hypothesize that the SIP signaling pathway is critical for GAS survival in human blood and systemic disease pathogenesis. To test this hypothesis, we first assessed whether the SIP signaling pathway controls SpeB biogenesis during growth in human blood ex vivo. To test this hypothesis, the isogenic SIP* mutant strain was grown to late exponential phase (A600, ∼1.5) in THY, washed with sterile PBS, and suspended in an equal volume of fresh human blood ex vivo. Consistent with our observations with GAS growth in saliva, the addition of synthetic SIP caused robust induction of speB expression in the SIP* mutant during growth ex vivo in blood (Fig. 5A). Restoration of speB expression in the SIP* mutant was specific for the amino acid sequence of inferred native SIP, as the scrambled peptide (SCRA) failed to induce speB expression (Fig. 5A). Collectively, these data indicate that SIP signaling is responsible for the induction of speB expression during GAS growth in blood ex vivo.
SIP signaling occurs during GAS growth in human blood and contributes significantly to GAS growth in human blood. (A) Addition of synthetic SIP restores speB expression in the isogenic SIP* mutant strain during growth in human blood. The fold change in speB transcript levels relative to unsupplemented bacterial growth is shown. The data graphed are means ± standard deviations for three biological replicates. (B) Bactericidal assay of the indicated GAS strains. Bacteria were grown to mid-exponential phase (A600, ∼0.4) in THY, and approximately 100 CFU of each GAS strain was inoculated into 300 μl of fresh human blood. After 3 h of incubation at 37°C, the multiplication factors were calculated by dividing the numbers of CFU per milliliter obtained after 3 h of incubation by the starting inoculum. The P values (n.s., not significant [P > 0.5]; ****, P < 0.0001) for the indicated strains were determined by comparison to WT GAS. The data graphed are means ± standard deviations for three biological replicates.
Next, we assessed the contribution of the SIP signaling pathway to GAS survival in human blood. The parental WT and isogenic mutant GAS strains were grown in human blood for 3 h, and bacterial growth was assessed by counting the number of CFU. Compared to the WT strain, the ΔropB, ΔspeB, and SIP* mutant strains were defective in their ability to proliferate in blood (Fig. 5B). However, the WT-like growth phenotype in blood was restored in the SIP* mutant strain complemented with pDC-SIP, suggesting that provision of SIP in trans is sufficient to reverse the defective phenotype of the SIP* mutant (Fig. 5B). Together, these results suggest that SIP-dependent gene regulation is crucial for GAS survival ex vivo in human blood and may contribute to GAS virulence during systemic infection.
SIP-dependent speB expression contributes to GAS resistance against LL37-mediated cytotoxicity.Antimicrobial peptides such as LL37 are abundant in human saliva and blood. LL37 mediates its cytotoxicity by inducing bacterial membrane lysis (40–43). Given that SIP-dependent gene regulation is crucial for GAS survival in human saliva and blood ex vivo, we hypothesized that the SIP signaling pathway contributes to GAS resistance against LL37-induced cytotoxicity. To test this hypothesis, GAS grown to stationary phase was washed and incubated with LL37 in phosphate buffer for 45 min. The bacterial resistance to LL37-mediated cytotoxicity was assessed by comparing the survival of untreated and LL37-treated GAS. Consistent with the role of SpeB as an LL37-degrading protease (41, 44), the SpeB-producing WT and trans-complemented GAS strains exhibited significant resistance against LL37 treatment (Fig. 6). Conversely, the isogenic ΔspeB, ΔropB, and SIP* mutant strains, which cannot produce SpeB, were killed significantly more efficiently by LL37 than the SpeB-producing strains (Fig. 6) (P < 0.005). These data indicate that the resistance to LL37-mediated cytotoxicity conferred by SIP-dependent speB expression contributes to GAS survival.
SIP-dependent gene regulation confers GAS resistance against LL37-mediated cytotoxicity. GAS strains grown to stationary phase of growth in THY (A600, ∼1.7) were washed with PBS and incubated with 2.5 μM synthetic LL37 in phosphate buffer for 45 min at 37°C. The antimicrobial effect of LL37 was assessed by calculating the ratio of the number of surviving cells in the LL37-treated group to the total number of bacteria incubated in the mock-treated group. Statistical significance was determined by t test. P values (n.s., not significant [P > 0.5]; ****, P < 0.0001) for the indicated strains were determined by comparison to the WT.
The SIP signaling mechanism is conserved among diverse GAS emm types.Genes encoding RopB, SIP, and SpeB are highly conserved among emm types, suggesting that the SIP signaling pathway is a conserved virulence regulatory mechanism among genetically diverse strains (4). To test the hypothesis that the SIP signaling mechanism is functional in various GAS emm types, we performed a synthetic SIP addition experiment using genetically diverse GAS strains that belong to M-protein serotypes M1, M3, M12, M59, and M89 (45–49). The genomes of the strains used have been sequenced and have WT alleles for all known major regulatory genes, including ropB, mga, and covRS. The strains of all tested emm types encode the same inferred SIP amino acid sequence.
Expression of speB occurs predominantly during the stationary phase of GAS growth in THY medium (4, 23, 25, 50). Thus, we tested whether the addition of the synthetic peptide containing the amino acid sequence of SIP decouples the growth phase dependency of speB expression and causes the early onset of speB expression in these different strains. Consistent with our previous observations in the GAS M3 serotype (4), SIP-specific induction of speB expression occurred during the exponential phase of growth in all tested strains (Fig. 7). Thus, SIP-dependent upregulation of speB expression is a conserved regulatory mechanism among diverse GAS emm types commonly causing human infections.
The SIP signaling pathway is conserved among several GAS M-protein serotypes. WT GAS isolates belonging to M-protein serotypes M1 (MGAS2221), M3 (MGAS10870), M59 (MGAS15249), M89 (MGAS26844), and M12 (MGAS9429) were grown to mid-exponential phase (A600, ∼0.4). Cells were subsequently incubated with either 500 nM synthetic SIP or scrambled peptide (SCRA) for an additional 60 min. The transcript levels of speB were assessed by qRT-PCR, and the fold change in speB expression relative to that in DMSO-supplemented growth is shown.
SIP is produced in vivo in infected humans.Next, we tested the hypothesis that SIP is produced during human infection and evokes production of anti-SIP antibodies by measuring anti-SIP antibody titers by enzyme-linked immunosorbent assay (ELISA). A random 9-amino-acid peptide derived from an unrelated GAS protein was used as a nonspecific control. Serum samples from 5 convalescing patients with previous invasive GAS infections and 5 pediatric patients with culture-positive GAS pharyngitis were assessed. All tested serum samples from patients with either invasive or pharyngeal GAS infections had anti-SIP antibody titers of a 1:1,000 dilution. However, even at a 1:100 dilution, the serum samples failed to react with the nonspecific control peptide. Collectively, the human serologic data provide evidence that SIP is produced in vivo in infected humans.
DISCUSSION
The antivirulence approach is an emerging paradigm to combat bacterial infections by targeting either the virulence factors or regulatory networks controlling virulence factor production for antimicrobial development (51–53). The ideal target for an antivirulence strategy must be active during infection and during the time of treatment and participate in disease pathogenesis (51–53). Thus, knowledge of the spatiotemporal pattern of virulence factor production in vivo is a crucial prerequisite for successful antivirulence targeting studies. Given that the SIP signaling pathway is active in the host and that SIP-mediated SpeB production is critical for GAS virulence in invasive animal models of infection (4), the SIP signaling pathway is an attractive target for antivirulence strategies to treat GAS infections. However, the contribution of the SIP signaling pathway to GAS survival in the oropharynx, the primary route of GAS entry into the host, remains unknown. In this study, we demonstrated that the SIP signaling pathway is the primary regulatory mechanism controlling speB expression during GAS growth ex vivo in saliva. Importantly, SIP-mediated speB regulation is critical for bacterial survival in human saliva and contributes significantly to oropharyngeal GAS colonization. Collectively, our results suggest that the SIP signaling pathway is active during GAS infection and participates in disease pathogenesis in multiple host niches, including the oropharynx (4, 23).
GAS employs a temporal pattern of speB expression during growth ex vivo in saliva that is distinct from the kinetics of the speB transcript profile during GAS growth in vitro (Fig. 2A and B). GAS triggers a transient but robust induction of speB expression during the early stages of growth in saliva (Fig. 2B), resulting in the accumulation of large amounts of extracellular SpeB protease (Fig. 2C). Early induction of SpeB protease production in saliva is likely an adaptive strategy to achieve sufficient quantities of SpeB protease in saliva before the onset of host innate immune responses. Given that human saliva contains several antimicrobial mechanisms (29, 40), the protease activity of SpeB may aid GAS survival in saliva by negating the cytotoxicity of host immune factors. Consistent with this, SIP-dependent SpeB production aids GAS survival in the presence of LL37 (Fig. 6), indicating that SpeB-mediated cleavage of LL37 likely contributes to GAS evasion of LL37-mediated cytotoxicity.
The temporal pattern of SpeB biogenesis during GAS growth in human saliva also has significant implications for future translational studies targeting the SIP signaling pathway for antimicrobial development. SIP signaling occurs only during the early stages of GAS growth ex vivo in saliva and is relatively inactive during the later stages of growth in saliva (Fig. 2B). Thus, the therapeutic targeting of the SIP signaling pathway to treat oropharyngeal GAS infections may be more effective during early stages of GAS infection. Alternatively, prophylactic targeting of the SIP signaling pathway may be a viable option, as anti-SIP antibodies are likely to be disruptive to SIP signaling and SpeB production. Furthermore, SpeB is present in relative abundance for a prolonged period during GAS growth ex vivo in saliva. Importantly, SpeB-producing strains demonstrated a better ability to persist in saliva and colonize the mouse oropharynx. Thus, targeting SpeB for antimicrobial development may be an effective strategy to treat oropharyngeal GAS infections.
GAS reaches a maximum population density of only 107 CFU/ml, which is 2 log10-fold less than the bacterial population density observed during GAS growth in vitro (Fig. 2A and B). Our observations are consistent with the GAS population densities reported in prior ex vivo growth studies in saliva or the GAS burden during acute pharyngitis in human patients (30–33). During growth in vitro, the SIP-mediated upregulation of speB expression occurs only at population densities greater than 108 CFU/ml (Fig. 2B). Although GAS does not reach such high population densities during growth in human saliva, it has the ability to induce speB expression at significantly lower population densities (∼106 CFU/ml) (Fig. 2A). These results suggest that additional mechanisms or signals in saliva may contribute to the upregulation of speB expression. To address the niche-specific distinction in the regulation of speB expression in saliva, we considered two possibilities. First, the host uses salivary glycoproteins to promote GAS aggregation as a defense mechanism to prevent bacterial attachment to epithelial surfaces and eliminate GAS from the oral cavity (54). Thus, it is possible that GAS may reach locally high bacterial population densities within a confined environment, such as bacterial aggregates, which leads to the local accumulation of SIP and induction of speB expression. Consistent with this, Staphylococcus aureus at a relatively low population density within a confined space, such as phagosomes, senses the accumulating extracellular peptide signal and activates the quorum sensing regulon (55, 56). Alternatively, additional bacterial or host-derived molecules present in saliva may act as inducers that provide the initial impetus toward the activation of the SIP signaling pathway, which leads to the autoinduction of the endogenous SIP signaling pathway and upregulation of speB expression. In accordance with this, Pseudomonas aeruginosa uses the excess aromatic amino acids in the sputum from cystic fibrosis patients as precursors for the synthesis of a quorum sensing signal molecule and activates the quorum sensing regulon (57). Additional investigations are required to elucidate the mechanistic basis for speB expression at relatively low GAS population densities in saliva.
The GAS genome encodes four different peptide-signaling systems comprised of sensor-peptide pairs, namely, ComR-ComS, SilAB-SilCR, Rgg-SHP, and RopB-SIP (4, 58–60). The ComRS system is involved in the regulation of genetic competence in streptococci, but its role in GAS remains elusive (61, 62). Although the SilCR signaling pathway participates in GAS pathogenesis, it is not conserved among GAS emm serotypes and is encoded by less than 25% of the sequenced GAS genomes (59, 60). The Rgg-SHP quorum sensing system is conserved among GAS emm serotypes, but its contribution to GAS pathogenesis is not fully understood (58, 59). In contrast, the RopB-SIP signaling pathway is highly conserved in all the sequenced GAS genomes, is functional in diverse GAS emm serotypes, and is critical for GAS disease pathogenesis in multiple host anatomic sites (4, 22) (Fig. 7). These properties of the SIP signaling pathway, combined with the observed immunological recognition of SIP by the host immune system, make the SIP signaling pathway a potential candidate for GAS vaccine development.
In conclusion, we demonstrate that GAS employs a combination of early activation of the SIP signaling pathway and timely SpeB production for successful persistence in saliva and colonization of the mouse oropharynx. The critical differences in the SIP signaling profile observed between GAS growth in vitro and ex vivo also underscore the significance of the need to study bacterial virulence regulatory programs under conditions that simulate their natural environment. In summary, in addition to elucidating the molecular details of niche-specific virulence regulation by the SIP signaling pathway, the results from this study also provide the scaffold for future translational studies targeting the SIP signaling pathway to treat or prevent pharyngeal GAS infections.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions.The bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. Strain MGAS10870 is a previously described invasive serotype M3 isolate whose genome has been fully sequenced (46). MGAS10870 is representative of serotype M3 strains that cause invasive infections and has wild-type sequences for all known major regulatory genes (46). The Escherichia coli DH5α strain was used as the host for plasmid constructions. GAS was grown routinely on Trypticase soy agar containing 5% sheep blood (BSA; Becton Dickinson) or in Todd-Hewitt broth containing 0.2% (wt/vol) yeast extract (THY; Difco). When required, chloramphenicol was added to a final concentration of 5 μg/ml. All GAS growth experiments were done in triplicate on three separate occasions for a total of nine replicates. Overnight cultures were inoculated into fresh medium to achieve an initial absorption at 600 nm (A600) of 0.03. Bacterial growth was monitored by measuring the A600. The E. coli strain was grown in Luria-Bertani (LB) broth (Fisher).
Collection of human saliva.Saliva from adult volunteers was collected on ice under a protocol approved by the Institutional Review Board at Houston Methodist Research Institute (approval number Pro00003833) using the method described previously with minor modifications (30). Dithiothreitol (DTT; Gold Biotechnology) was added at a final concentration of 2.5 mmol to the saliva pool, and the mixture was incubated on ice for 30 min. The saliva was clarified by centrifugation at 23,000 × g for 1 h, followed by filtration through a 0.22-μm-pore-size membrane filter (Corning, NY). Pooled saliva was stored frozen at −20°C. Saliva from at least four donors was pooled to minimize the potential effects of donor variation.
GAS growth in human saliva.The ability of GAS strains to grow and persist in human saliva was evaluated as described previously (30). Briefly, human saliva was collected from healthy volunteers and pooled as described above. GAS was grown overnight in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY; BD Biosciences, Sparks, MD), diluted 1:100 with fresh THY, and grown to the growth phase indicated above. The bacterial cells were pelleted, washed twice with sterile PBS, and suspended in saliva at ∼1 × 105 CFU/ml. Aliquots were removed at the time points indicated above and in the figures. Samples were serially diluted 10-fold in sterile PBS and plated in duplicate on Trypticase soy agar plates supplemented with 5% sheep blood (BD Biosciences). The plates were incubated overnight, and colonies were counted to determine the number of CFU. All incubations were at 37°C with 5% CO2. Each experiment was performed in triplicate on three separate occasions.
Mouse oropharyngeal infection.All animal experiments were conducted under a protocol approved by the Houston Methodist Research Institute Institutional Animal Care and Use Committee (approval number AUP-1215-0069). Twenty 3- to 4-week-old female CD1 mice (Harlan Laboratories) were inoculated intranasally with 2 × 108 CFU of the appropriate GAS strain in 50 μl phosphate-buffered saline (PBS). Mouse throats were swabbed prior to inoculation to ensure the absence of beta-hemolytic bacteria and daily thereafter for a total of 7 days to assess GAS colonization. Throat swab specimens were vortexed in 300 μl sterile PBS at 1,000 rpm on a high-speed microplate shaker (Illumina, San Diego, CA), and the numbers of CFU per milliliter were determined by serial diluting 1:10 in PBS, plated on group A streptococcal selective agar with 5% sheep blood (SSA; Becton Dickinson), and grown overnight at 37°C, and the beta-hemolytic colonies were counted.
GAS growth studies in human blood.Whole blood was drawn from consenting, healthy, nonimmune donors in sodium heparin tubes (Becton Dickinson) under a Houston Methodist Research Institute Institutional Review Board-approved experimental protocol (approval number Pro00004933). GAS growth in blood was performed as described previously (63). Indicated GAS strains were grown in THY at 37°C in 5% CO2 to mid-exponential phase (A600, ∼0.4) and harvested. The cells were washed twice and suspended in an equal volume of sterile PBS. Approximately 20 to 100 CFU of each GAS strain was inoculated into 300 μl of fresh human blood. Samples were incubated for 3 h at 37°C in 5% CO2 with end-to-end rotation. The numbers of CFU per milliliter were determined by serially diluting the samples 1:10 in PBS, plating the samples, and growing the samples overnight at 37°C in 5% CO2, and the beta-hemolytic colonies were counted. Multiplication factors were calculated by dividing the number of CFU per milliliter determined after 3 h of incubation by the starting inoculum. Each experiment was performed in triplicate on separate occasions.
GAS RNA isolation and gene transcript analysis from human saliva and blood.One volume of GAS strains grown in human saliva or human blood was collected in 2 volumes of RNAprotect (Qiagen) at the time points indicated above, incubated at room temperature for 10 min, and harvested by centrifugation. Bacterial cell pellets grown in blood were suspended in 10 volumes of ammonium chloride lysing solution (Becton Dickinson), incubated for 10 min on ice, and separated from lysed erythrocytes by centrifugation at 3,000 × g at 4°C for 10 min. RNA was isolated from the GAS growth in saliva or blood and purified using an RNeasy kit (Qiagen) according to the manufacturer's instructions. A260/A280 ratios were used to assess RNA integrity. cDNA was synthesized from purified RNA using SuperScript III reverse transcriptase (Invitrogen). TaqMan PCR was performed with an ABI 7500 Fast system (Applied Biosystems). Comparison of transcript levels was performed by the ΔCT threshold cycle (CT) method of analysis using tufA as the endogenous control gene (64). The sequences of the probes and primers used in the TaqMan PCR are listed in Table S2.
Western immunoblot analysis.MGAS10870 was grown in human saliva to the time points indicated above, and supernatant was collected by centrifugation. The culture supernatant was filtered through a 0.22-μm-pore-size filter (Millipore), and the filtrate was concentrated by drying with a Speed-Vac. Equal volumes of concentrated supernatant sample were resolved on a 15% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Bio-Rad), and probed with polyclonal anti-SpeB rabbit antibodies. Secondary antibody conjugated with horseradish peroxide was used to detect SpeB and visualized with chemiluminescence using a SuperSignal West Pico rabbit IgG detection kit (Thermo Scientific).
Synthetic peptide addition assay during GAS growth in human saliva and human blood.Synthetic peptides of high purity (>90% purity) obtained from Peptide 2.0 (Chantilly, VA) were suspended in 100% dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Stock solutions were aliquoted and stored at −20°C until use. Working stocks were prepared by diluting the stock solution in 25% DMSO.
LL37 cytotoxicity assays.GAS strains were grown to stationary phase in THY (A600, ∼1.7), washed with PBS, and suspended in 10 mM sodium phosphate buffer (pH 6.8). Similar starting numbers of CFU per milliliter of each strain were preincubated at 37°C for an additional 2 h. Subsequently, LL37 was added to a final concentration of 2.5 μM, the culture was incubated for 45 min, and bacterial killing was assessed by counting the numbers of CFU per milliliter. The LL37 cytotoxicity was assessed by calculating the ratio of the number of surviving cells in the LL37-treated group to the number of bacteria incubated in the mock-treated group.
Analysis of in vivo SIP expression by ELISA.ELISA was used to determine if SIP is expressed during human GAS infection. Synthetic SIP was used to coat MaxiSorp ELISA plates (Nunc) at 0.5 μg/well at 4°C overnight. A synthetic 9-mer peptide derived from an unrelated GAS cytosolic protein (CvfA, SpyM3_1376) with an amino acid sequence of YALISIRLK was used as a nonspecific control. Convalescent-phase human serum samples were collected from 5 patients with previous invasive GAS infections under a protocol approved by the Houston Methodist Research Institute Institutional Review Board. Acute-phase human serum samples from 5 pediatric patients with acute GAS pharyngitis were also collected. A serial 2-fold dilution of the serum samples was used, and secondary horseradish peroxidase (HRP)-conjugated anti-human immunoglobulin antibody (Millipore Sigma Inc.) was used to detect bound primary antibody. The absorbance of the plates at 420 nm (A420) was read. An A420 reading greater than at least thrice the value of the PBS negative control was considered a positive reaction.
Statistical analysis.Repeated-measure analysis of variance (ANOVA) was used to test the differences in nasopharyngeal colonization between strains. Paired Student's t test was used to compare the ability of GAS strains to survive in human saliva or blood by comparing the log10 number of CFU per milliliter of the indicated strains on day 6 or multiplication factors after 3 h of incubation, respectively. Statistical significance was assigned at a two-sided P value of 0.05, using Bonferroni's adjustment for multiple comparisons when appropriate. GraphPad Prism software was used for statistical calculations.
ACKNOWLEDGMENTS
This work was supported by funds from the Fondren Foundation to J.M.M. and National Institutes of Health grant 1R01AI109096-01A1 to M.K. H.D. was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2017R1A6A3A03008353).
We declare no conflict of interest.
FOOTNOTES
- Received 2 March 2018.
- Accepted 4 March 2018.
- Accepted manuscript posted online 12 March 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00169-18.
- Copyright © 2018 American Society for Microbiology.
