Deciphering the Regulatory Role and Molecular Interactions that Drive the Competence Regulon Quorum Sensing Circuitry in Streptococcus constellatus

Abstract

Streptococcus constellatus, a member of the Streptococcus anginosus group, is a Gram-positive bacterial species commonly found in the oral and gastrointestinal tracts; however, it can become pathogenic, particularly in immunocompromised individuals. S. constellatus is associated with the development of purulent infections that typically require a combination of antibiotic therapy and surgical intervention for effective treatment. This understudied emerging pathogen appears to possess comABCDE quorum sensing (QS) circuitry, which is common in many streptococci species, however the molecular interactions that drive this circuitry and its regulatory function remain unexplored. Herein, we confirmed the identity of S. constellatus competence-stimulating peptide (CSP) and performed phenotypic assays to investigate its regulatory role in competence and biofilm development. To examine the role of each amino acid within the CSP sequence in QS activity, we conducted D-amino acid and alanine scans of the S. constellatus CSP. Given the uniquely high natural QS induction observed in S. constellatus, we developed a ΔcomC luciferase reporter system to assess the activity of the D-amino acid and alanine scan analogs on the competence regulon. Our results revealed that a single Alanine substitution could lead to a 60-fold increase in CSP potency. Furthermore, through circular dichroism analysis, we investigated the correlation between secondary structure and biological activity. Overall, this study aims to enhance our understanding of S. constellatus, a relatively understudied species with pathogenic potential, and aid in the development of strategies to mitigate its pathogenicity.

Graphical Abstract

Introduction:

The Streptococcus anginosus group (SAG) is a group of Gram-positive streptococci and a subgroup of viridans streptococci.^1,2 It comprises three distinct species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus.^2,3 Although bacterial species of this group naturally inhabit the human body, primarily in the oral cavity, gastrointestinal tract, and urogenital region, they have increasingly emerged as pathogens in invasive infections.⁴ Of particular interest is S. constellatus, which has the potential to become pathogenic, leading to severe infections such as empyema and necrosis in organs like the lungs, liver, and brain, particularly in individuals with underlying health conditions.^5,6,7 The pathogenic risk of S. constellatus should not be overlooked, especially in patients with immunosuppression, as infection often requiring intensive treatment, potentially involving surgical intervention.⁷ Although these bacteria are recognized for their potential to cause challenging infections, the molecular mechanisms behind their pathogenicity remain unclear, as most research has been devoted to species identification rather than exploring their pathogenic behavior.^8,9

Bacterial competence is the process by which bacteria acquire and integrate external DNA into their genomes, playing a crucial role in both horizontal gene transfer (HGT) and genetic recombination.^10,11,12 While competence is vital for bacterial evolution and enhances genetic diversity, it raises clinical challenges as these bacteria can acquire antibiotic resistance genes, complicating treatment options.^13,14 Maintaining competence is energetically expensive for bacteria as they have to allocate resources away from other essential activities.^15,16 To minimize adverse effects and optimize transformation efficiency, the initiation and termination of competence are rigorously regulated.¹⁷ This regulatory process is governed by a well-established mechanism in streptococci known as quorum sensing (QS) that enables bacteria to detect their population density and modulate gene expression once a high population threshold is reached.^18,19,20 In both Gram-negative and Gram-positive bacteria, QS plays a role in regulating many group behavior phenotypes. In many streptococcal species, the QS circuit that governs competence, known as the comABCDE regulon, initiates a signaling cascade essential for competence development.²¹ Initially, the precursor peptide ComC undergoes modification and is secreted as a mature competence stimulating peptide (CSP) signal by the ABC transporter ComAB. Upon reaching the threshold concentration of CSP in the extracellular environment, the CSP binds and triggers the activation of the membrane-bound histidine-kinase receptor (ComD). As a result of this receptor binding, ComE, a cytoplasmic response regulator, is phosphorylated, subsequently serving as a transcription factor that activates the expression of the QS genes (comABCDE) and the alternative sigma factor gene (comX), the principal regulator of QS-regulated phenotypes such as competence (Figure 1)

Figure 1.

Illustration of the general CSP-mediated quorum sensing pathway in streptococci.

Although the native CSP sequence of S. constellatus was inferred from genomic data in prior studies, it had not been experimentally validated through isolation from bacterial supernatants. Sequencing data provides strong evidence that S. constellatus is naturally competent, and the addition of the predicted 16-amino acid synthetic CSP in S. constellatus has been shown to upregulate competence.²² However, the role of the competence regulon in modulating other phenotypic behaviors in S. constellatus is still unclear. This study aimed to verify the identity of the S. constellatus CSP through isolation from bacterial supernatants, investigate the regulatory role of the competence regulon in S. constellatus, and uncover the molecular interactions that govern CSP/ComD interactions and lead to activation of the QS pathway. To achieve our objective, we began by utilizing peptide extraction and tandem mass spectrometry to elucidate the structure of the S. constellatus CSP. We then carried out full D-amino acid and alanine scans of the S. constellatus CSP sequence to identify structural motifs essential for ComD binding and activation, following the development of a luciferase-based S. constellatus QS reporter strain to quantify the activity of each analog. We next utilized CD spectroscopy to determine the secondary structure and conformational properties of all D-amino acid and alanine-substituted S. constellatus CSP analogs. Finally, we conducted phenotypic assays to investigate the role of the S. constellatus competence regulon in regulating bacterial competence and biofilm formation.

Results and Discussion

Prediction and isolation of the S. constellatus CSP signal from cell-free supernatants

Distinct species in the Streptococcus anginosus group often encode and respond to the same CSPs, whereas in other groups of streptococci, CSPs are typically species-specific and, in some cases, specific to particular groups of strains.^22,23,24,25 Previously, the S. constellatus CSP was predicted using genomic data, followed by synthesis of a 16-amino acid peptide (DSRIRMGFDFSKLFGK), and demonstration that the synthetic peptide can induce competence in S. constellatus.²² We verified the presence of the same ComC sequence in our tested S. constellatus strain by performing PCR amplification and sequencing of the comC gene, resulting in a predicted CSP sequence that aligned with the previously reported 16-amino acid peptide (Figure 2).

Figure 2. Predicted S. constellatus CSP Sequence Derived from Sequencing Data.

Identification of S. constellatus CSP based on comC sequencing results.

Although Havarstein and co-workers have demonstrated that the mature CSP signal is generally obtained by processing ComC at a double glycine leader sequence,²² it has been reported before that ComC can undergo further modifications to generate a mature CSP signal that is not simply the sequence following the double glycine repeat, through cleavage by either an extracellular SepM-like protease or directly by the membrane-bound ComAB transporter.²⁶ We therefore sought to isolate the processed CSP from bacterial supernatants to verify that the predicted CSP sequence aligned with the mature CSP formed by ComC processing by ComAB. Unfortunately, we were initially unable to detect a peptide mass corresponding to the 16-mer CSP in the culture supernatant. One potential reason for the challenge in isolating CSP could be the low concentration by which the peptide is produced and its affinity to the cell surface, preventing it from diffusing freely into the medium. This could lead to reduced amounts of CSP in the supernatant, making it difficult to extract. Another possibility is that competence development in many streptococcal species, such as S. pneumoniae, relies on a mechanism involving direct cell-to-cell contact, where competence initiator cells transfer CSP to neighboring cells. This could also result in reducing the accumulation of CSP in the culture medium, as it would mainly be confined to areas where active and inactive cells are in direct contact rather than dispersing into the culture medium.²⁷ To address the challenge of CSP extraction, we artificially induced its production using an abiotic synthetic activator from our alanine scan analogs. After diluting the S. constellatus overnight culture, incubation continued until OD₆₀₀ reached 0.2, at which point CSP K16A was introduced. The supernatants from S. constellatus cultures were then treated with ammonium sulfate to isolate crude peptides, which were subsequently fractionated using RP-HPLC and masses that matched the predicted CSP were detected, indicating that the predicted 16-mer CSP is the mature peptide (Figure 3).

Figure 3.

Isolation and detection of the S. constellatus CSP from cell-free supernatants. The RP-HPLC chromatogram of total proteins isolated from the supernatant sample highlighting the fraction collected from 19 to 21 min (red). See the Supporting information for full experimental details.

Comparison of isolated and synthetic S. constellatus CSP:

The identity of the S. constellatus native CSP was further validated through the synthesis of its predicted peptide sequence. We then conducted a comparison between the purified synthetic peptide and the peptide extracted from S. constellatus cell-free supernatants using analytical HPLC. Both individual peptides displayed a dominant peak at the same retention time, and their combined fractions in equal proportions produced a single peak with the same retention time as the individual fractions (Figure 4C). The exact masses of both the synthetic and isolated peptides were measured and found to match the expected mass of the 16-amino acid CSP sequence, within a 5-ppm error (Figure 4B). Finally, through MS/MS analysis of the isolated CSP, the connectivity of the sequence was confirmed (Table S5). These findings together corroborate that the CSP sequence in S. constellatus is the 16-amino acid peptide, DSRIRMGFDFSKLFGK (Figure 4A)

Figure 4. Comparative Analysis of Purified Synthetic and Isolated S. constellatus CSPs.

(A) Proposed structural representation of the 16-amino acid S. constellatus CSP. (B) Exact mass analysis of synthetic and extracted peptides using ESI-TOF MS. (C) RP-HPLC chromatographic comparison of purified natural CSP, synthetic CSP, and their mixture.

Construction of S. constellatus luciferase QS reporter strain:

A luciferase-based S. constellatus ATCC 27823 QS reporter strain was developed to evaluate the ability of native S. constellatus CSP and its analogs to modulate the activity of the S. constellatus ComD receptor. This reporter strain (ATCC 27823 PcomX::luc) was developed following a strategy similar to that introduced by Salvadori et al., enabling direct quantification of comX expression upon CSP/ComD binding.^28,29,30,31 The successful construction of the reporter strain was confirmed by sequencing, showing the promoter for comX was inserted immediately upstream of the Luc gene. Luminescence assays conducted on the reporter strain (ATCC 27823 PcomX::luc) revealed luminescence when the S. constellatus reporter culture was treated with the native S. constellatus CSP signal and 15 μg/mL D-luciferin. Notably, luminescence was also observed in negative control samples (DMSO), indicating that sufficient amounts of endogenous CSP are naturally produced by S. constellatus to fully activate the QS circuitry (Figure S13C). To eliminate endogenous CSP production, which would interfere with the accurate evaluation of synthetic CSP analogs, we constructed a ΔcomC S. constellatus QS reporter strain using homologous recombination.³² The comC gene encodes the precursor for CSP, ComC, and its deletion prevents the synthesis of endogenous CSP, thereby ensuring that the luminescence response is exclusively due to the addition of external CSP or its analogs. The knockout reporter strain was verified by PCR and Sanger sequencing. Luminescence assays conducted on the ΔcomC reporter strain (ATCC 27823 ΔcomC PcomX::luc) revealed luminescence when the S. constellatus reporter culture was treated with the native S. constellatus CSP signal and 15 μg/mL D-luciferin. Importantly, luminescence was not observed in the DMSO negative control samples, indicating lack of endogenous CSP production by S. constellatus (Figure S13D).

Design and Synthesis of S. constellatus CSP alanine and D-amino acid scans:

To gain insight into the role of each sidechain residue and chiral center in the 16-amino acid S. constellatus CSP, we conducted a comprehensive alanine and D-amino acid scan of the CSP sequence. Each CSP analog was synthesized via Fmoc solid-phase peptide synthesis, employing 4-benzyloxbenzyl alcohol (Wang) resin as the solid support. Upon completion of on-resin synthesis for each alanine- or D-amino acid-substituted CSP, the peptides were cleaved from the support and purified to a homogeneous state using semi-preparative RP-HPLC. The activity of S. constellatus CSP analogs was then quantitatively assessed through a luciferase reporter gene assay using the developed S. constellatus ΔcomC reporter (ATCC 27823 ΔcomC PcomX::luc; See additional experimental details and peptide characterization in the Supporting Information).

SAR of the S. constellatus alanine scan analogs:

To evaluate the importance of each sidechain residue in the 16-amino acid S. constellatus CSP for QS activation, luciferase reporter gene assays were performed to measure the activity of alanine-substituted peptide analogs. Since this analysis was aimed at evaluating the role of the sidechain functionality on activity, glycine residues, who do not have sidechain residues, just a hydrogen as their sidechain, were not included in the analysis. The S. constellatus CSP can be divided into three distinct segments: the N-terminal region (first three amino acid residues), the central region (residues 4–13), and the C-terminal region (last three amino acid residues, residues 14–16). Alanine scanning of the N-terminal region highlighted the critical role of Asp1 in QS activation, as its substitution with alanine altered CSP activity, converting it into a competitive inhibitor. Alanine substitution at Ser2 had minimal effect on activity compared to the native CSP, while replacing Arg3 with alanine resulted in a complete loss of function, highlighting the essential role of the third residue. Our analysis of the N-terminal region of S. constellatus CSP is in line with previous work on CSPs from other streptococcal species belonging to the mitis group of streptococci, where the first negatively charged amino acid (Glu or Asp) was shown to be vital for QS activation and the third residue (Arg) crucial for CSP function, indicating a conserved motif at these positions.^33,34,35 The alanine substitution at Ile4 resulted in a significantly enhanced analog, with an approximately 60-fold increase in potency (EC₅₀ = 0.089 nM) compared to the native peptide (EC₅₀ = 5.2 nM) (Table 1). Of all alanine analogs, substitution of this Ile4 side chain exhibited the most significant improvement in potency, indicating that shortening the side chain probably enhanced receptor binding, likely through a reduction of steric clashes within the ComD receptor binding pocket, affording optimized peptide:receptor interactions, and eventually leading to QS activation at low peptide concentrations. Substituting alanine at Met6 or Ser11 resulted in analogs with only a slight increase in potency, showing a 2.5-fold decrease in EC₅₀ values compared to the native CSP (Table 1). On the other hand, substitutions at Arg5, Asp9, or Phe10 yielded peptides with activities similar to the native CSP, suggesting that these residues are not important for QS activation. In contrast, replacing Lys12 with alanine led to a peptide with reduced activity, with an EC₅₀ value 16-fold higher (EC₅₀ = 82), highlighting the importance of Lys12 for QS activation. Additionally, the Phe8 substitution reduced activity by 3-fold, indicating a moderate effect of this residue on receptor activation. Finally, substitutions at Leu13 and Phe14 abolished QS activation, likely through lack of receptor activation, indicating the likely crucial role of these residues in receptor binding. In contrast, the K16A substitution slightly improved potency, reflected by a lower EC₅₀ value. These results highlight that the C-terminal region in S. constellatus is essential for QS activation, as demonstrated by the complete loss of function in CSP F14A (Table 1). These results are unique to S. constellatus, as in other streptococcal CSPs the C-terminus was generally found to be dispensable.

Table 1.

EC50 or IC50 Values of Alanine-Screen Analogs against the ComD Receptor^[a]

Peptide Name	Peptide sequence	EC50 or IC50* (nM)[b]	95% CI[c]
Native CSP	DSRIRMGFDFSKLFGK	5.2	3.0 – 8.9
CSP D1A	ASRIRMGFDFSKLFGK	120*	75 – 200*
CSP S2A	DARIRMGFDFSKLFGK	5.6	2.5 – 12
CSP R3A	DSAIRMGFDFSKLFGK	----[d]	----
CSP I4A	DSRARMGFDFSKLFGK	0.089	0.046 – 0.17
CSP R5A	DSRIAMGFDFSKLFGK	4.8	3.7 – 6.3
CSP M6A	DSRIRAGFDFSKLFGK	1.9	1.1 – 3.2
CSP F8A	DSRIRMGADFSKLFGK	15	7.9 – 29
CSP D9A	DSRIRMGFAFSKLFGK	6.7	4.2 – 11
CSP F10A	DSRIRMGFDASKLFGK	6.4	4.5 – 8.9
CSP S11A	DSRIRMGFDFAKLFGK	2.0	1.1 – 3.2
CSP K12A	DSRIRMGFDFSALFGK	82	36 – 180
CSP L13A	DSRIRMGFDFSKAFGK	----[d]	----
CSP F14A	DSRIRMGFDFSKLAGK	----[d]	----
CSP K16A	DSRIRMGFDFSKLFGA	2.5	1.1 – 5.8

^[a]Refer to the Materials and Methods section or the Supporting Information for experimental procedures. Details on the reporter strain and dose-response curves for agonism and antagonism can be found in the Supporting Information. All assays were conducted in triplicate.

^[b]EC₅₀ or IC₅₀ values were determined by testing peptides over a broad concentration range.

^[c]Represents the 95% confidence interval.

^[d]EC₅₀ was not determined due to the analog’s low induction in initial agonism screening assays. Additional details are available in the Supporting Information

SAR of the S. constellatus D-amino acid scan analogs:

We began our D-amino acid scan analysis by focusing on the N-terminal region, where substitution of any of the first three positions were found to result in analogs with activities similar to the native CSP (Table 2). Collating these results with findings from our alanine screening, it is clear that for the N-terminal region the identity of the side chain is more important for biological activity than its orientation. In the central region of S. constellatus CSP, D-amino acid substitutions at Ile4, Arg5, Met6, or Leu13 led to a substantial decrease in potency with an increase in EC₅₀ values, ranging from 5- to 9-fold compared to the native CSP. With the exception of Leu13 where alanine substitution completely abolished activity, these results suggest that these positions are more sensitive to side chain orientation changes than sidechain modifications. In contrast, D-amino acid substitutions at Phe8, Asp9, Phe10, Ser11, or Lys12 resulted in EC₅₀ values comparable to or slightly lower than the native peptide, suggesting that side chain orientation is not a major determinant of activity (Table 2). This trend was consistent with the alanine substitutions at these positions, with the exception of Phe8 and Lys12, where the EC₅₀ values increased compared to the native CSP (3-fold and 16-fold, respectively). Finally, The D-amino acid substitutions at Phe14 and Lys16 resulted in approximately a two-fold reduction in the EC₅₀ value compared to the native CSP, although this change was not statistically significant (Table 2).

Table 2.

EC50 or IC50 Values of D amino acid Analogs against the ComD Receptor^[a]

Peptide Name	Peptide sequence	EC50 (nM)[b]	95% CI[c]
Native CSP	DSRIRMGFDFSKLFGK	5.2	3.0 – 8.9
CSP-d1	dSRIRMGFDFSKLFGK	9.7	4.3 – 22
CSP-s2	DsRIRMGFDFSKLFGK	7.9	4.0 – 16
CSP-r3	DSrIRMGFDFSKLFGK	13	4.0 – 46
CSP-i4	DSRiRMGFDFSKLFGK	25	12 – 54
CSP-r5	DSRIrMGFDFSKLFGK	47	20. – 110
CSP-m6	DSRIRmGFDFSKLFGK	35	24 – 51
CSP-f8	DSRIRMGfDFSKLFGK	4.9	4.3 – 5.7
CSP-d9	DSRIRMGFdFSKLFGK	3.3	1.8 – 6.3
CSP-f10	DSRIRMGFDfSKLFGK	4.7	2.6 – 8.3
CSP-s11	DSRIRMGFDFsKLFGK	1.8	1.5 – 2.1
CSP-k12	DSRIRMGFDFSkLFGK	3.7	2.6 – 5.1
CSP-l13	DSRIRMGFDFSKlFGK	25	18 – 35
CSP-f14	DSRIRMGFDFSKLfGK	2.0	1.1 – 3.7
CSP-k16	DSRIRMGFDFSKLFGk	1.7	0.70 – 4.3

^[a]Refer to the Materials and Methods section or the Supporting Information for experimental procedures. Details on the reporter strain and dose-response curves for agonism and antagonism can be found in the Supporting Information. All assays were conducted in triplicate.

^[b]EC₅₀ values were determined by testing peptides over a broad concentration range.

^[c]Represents the 95% confidence interval.

Structural analysis using CD spectroscopy:

To further understand how different modifications influenced the overall peptide structure and its correlation with bioactivity, we set out to analyze the structure of the native S. constellatus CSP and its D-amino acid and alanine scan analogs using CD spectroscopy. We assessed all synthetic S. constellatus CSP analogs in both aqueous conditions (PBS buffer, pH 7.4) (Figures S4 and S6) and membrane-mimicking conditions (20% trifluoroethanol in PBS, pH 7.4; Figures S5 and S7). Under both aqueous and membrane-mimicking environments, the native S. constellatus CSP displayed a random coil conformation, indicating a lack of defined structure. All D-amino acid scan analogs exhibited an unstructured, random coil conformation in both aqueous and membrane-mimicking conditions, similar to the native pheromone. Although most alanine scan analogs followed the same pattern, we observed a few exceptions. Among the alanine substitutions, substituting Arg3 with alanine was the only modification that led to a spectral pattern characteristic of a β-sheet and a loss of biological activity, emphasizing the importance of the Arg3 residue in both structural and biological function. In contrast, D9A (10.96% helicity) was the only alanine scan analog to exhibit helicity. Although this analog exhibited a promising EC₅₀ value, its bioactivity did not differ significantly from that of the native CSP. Alpha-helical structures are commonly associated with high biological activity of CSPs in many Streptococcal species, however the peptides in our study demonstrated significant bioactivity without displaying α-helix characteristics. Overall, no distinct correlation between helicity and bioactivity was observed in the peptides examined.

Phenotypic Assays:

Competence:

Given that the competence regulon QS pathway has been extensively studied and shown to regulate competence and biofilm formation in several Streptococcus species,^34,36,14 we sought to determine whether these mechanisms are similarly QS-dependent in S. constellatus. We initiated our phenotypic analysis by examining competence induction using an antibiotic resistance transformation assay. A spectinomycin resistance plasmid pDL278 (SpecR) was introduced into both wild-type and ΔcomC S. constellatus ATCC 27823 strains. We initially conducted transformation assays on the wild-type strain without exogenous CSP to further validate our findings that S. constellatus is naturally competent, and as expected, we observed many transformants on the agar plate following incubation (Figure 5A). We then assessed the effects of S. constellatus native CSP, S. constellatus CSP I4A (the highly potent activator identified in this study), and S. constellatus CSP D1A (the leading inhibitor) on competence induction of the wild-type strain (Figure 5B–5E). Corroborating our luminescence rereport gene assay results, incubation with a high concentration (10,000 nM) of the native CSP or the synthetic lead activator, CSP I4A, resulted in similar number of transformants to that of the DMSO control (without exogenous CSP addition), indicating that even without the addition of exogenous activator, the competence regulon QS circuitry in S. constellatus is activated to the maximum extent (Figure 5B–5C). Importantly, the addition of our lead inhibitor, CSP D1A, either alone or in combination with the native CSP, resulted in a significant reduction in the number of transformants, confirming that CSP D1A can effectively block competence induction (Figure 5D–5E). Next, we performed the same assay on the ΔcomC strain, initially without the addition of exogenous CSP, and did not observe any transformants, further validating the crucial role of comC in competence regulation (Figure 6A). We further performed a transformation assay using the ΔcomC reporter strain in the presence of varying concentrations of exogenous S. constellatus native CSP (10,000, 1,000, 100, and 20 nM) (Figure 6B–6E) and CSP I4A (10,000, 1,000, 100, and 0.4 nM) (Figure S11). Our results revealed transformation dependency on exogenous CSP addition in a dose-dependent manner, with lower CSP concentrations lead to corresponding reductions in the number of transformants.

Figure 5. Transformation assay of wild-type S. constellatus ATCC 27823.

The ability of wild-type S. constellatus ATCC 27823 to internalize a spectinomycin-resistance plasmid (pDL278, SpecR) was evaluated under different conditions: A) DMSO (no exogenous synthetic CSP); B) S. constellatus CSP (10,000 nM); C) S. constellatus CSP I4A (10,000 nM); D) S. constellatus CSP D1A (700 nM); and E) S. constellatus CSP D1A (700 nM) + CSP (20 nM). In all treatments, the amount of plasmid used was 200 ng. The number of transformants in each condition was assessed to evaluate the impact of each CSP analogs on plasmid uptake. See the Materials and Methods section for full experimental details and the Supporting Information for additional treatment conditions.

Figure 6. Transformation assay of S. constellatus ATCC 27823 ΔcomC.

The ability of S. constellatus ATCC 27823 ΔcomC to internalize a spectinomycin-resistance plasmid (pDL278, SpecR) was evaluated under different conditions and exhibits a dose-dependent response to CSP concentration: A) DMSO (no exogenous synthetic CSP); B) S. constellatus CSP (10,000 nM) C) S. constellatus CSP (1,000 nM); D) S. constellatus CSP (100 nM); E) S. constellatus CSP (20 nM). In all treatments, the amount of plasmid used was 200 ng. The number of transformants in each condition was assessed to evaluate the impact of exogenous CSP addition on plasmid uptake. See the Materials and Methods section for full experimental details and the Supporting Information for additional treatment conditions.

Biofilm Formation:

Considering that the competence regulon has been identified in other streptococci as a regulator of biofilm formation,^{36,14,37,38,39,40,41} a characteristic closely associated with increased pathogenicity, we aimed to investigate whether biofilm formation in S. constellatus is similarly governed by QS. To this end, we carried out a crystal violet assay to quantify biofilm formation in both the wild-type and the ΔcomC S. constellatus strains with each strain treated with the highest concentration of native CSP (10,000 nM) or DMSO (Figure 7). Our results exhibited no significant difference in biofilm production between the strains, whether treated with the highest concentration of CSP or without CSP, suggesting that the competence regulon does not affect biofilm formation.

Figure 7:

Biofilm formation by S. constellatus ATCC 27823 ΔcomC and wild-type strains was evaluated in the presence of exogenous 10,000 nM CSP and in the absence of CSP. No significant differences in biofilm formation were observed between the two strains, with biofilm production being comparable under both conditions.

Conclusion:

In this study, we confirmed the identity of the S. constellatus CSP to be a 16-amino acid peptide, DSRIRMGFDFSKLFGK, and demonstrated that this peptide plays a key role in regulating bacterial competence by activating the competence regulon, but appears to play no role in biofilm formation, generally a well-documented QS regulated phenotype. Significant endogenous CSP production was detected in S. constellatus by utilizing a luminescence reporter strain developed in this study. To eliminate interference from endogenous CSP production, a ΔcomC S. constellatus reporter strain was constructed, ensuring that luminescence responses are exclusively due to the addition of external CSP analogs. The ΔcomC S. constellatus reporter strain was then utilized to study the structure-activity relationship of the CSP in QS activation, likely through binding and activating the ComD receptor. Our findings highlight the vital role of the N-terminal region in QS activation, aligning with previous research on other streptococcal species, where mutation of the first negatively charged residue to alanine led to a QS inhibitor. Additionally, through alanine screening of the S. constellatus CSP, we identified a highly potent QS activator, CSP I4A, exhibiting an EC₅₀ value of 0.089 nM, about 60-fold more potent than the native CSP pheromone. Moreover, our results suggest that the C-terminal region of S. constellatus is crucial for QS activation, in contrast to other streptococcal species where the C-terminal region primarily influences peptide solubility and is generally dispensable. This study thus provides useful insights into designing effective CSP analogs and emphasizes the importance of specific residues for modulating QS activity in S. constellatus. Lastly, transformation assays with both wild-type and ΔcomC S. constellatus strains revealed that transformation is dependent on CSP in a dose-dependent manner, and that naturally produced CSP is sufficient to fully activate the competence regulon in S. constellatus resulting in maximum transformation efficiency without exogenous addition of synthetic CSP. Overall, this research expands our understanding of S. constellatus, a species with pathogenic potential but limited research, and offers valuable insights into further studies focused on attenuating its pathogenicity.

Material and methods:

Chemical Reagents and Instrumentation:

All chemical reagents and solvents used in this study were purchased from Chem-Impex, Aapptec, or Sigma-Aldrich and were used directly without any further purification. The 18 MΩ water was purified using a Barnstead Smart2Pure Pro water purification system. Reversed-phase high-performance liquid chromatography (RP-HPLC) was carried out using a Shimadzu system featuring a CBM-20A communications bus module, an SPD-20A UV/VIS detector, a CTO-20A column oven, two LC-20AT pumps, an SIL-20A autosampler, and an FRC-10A fraction collector. For the RP-HPLC analysis, solvents including 18 MΩ water and HPLC-grade acetonitrile (ACN) contained 0.1% trifluoroacetic acid (TFA). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data were collected using a Bruker Microflex spectrometer with a 60 Hz nitrogen laser and a Shimadzu MALDI-8020 spectrometer equipped with a 355 nm laser operating at 200 Hz. Both instruments were operated in reflectron positive ion mode, with acceleration voltages of 19.01 kV for the Bruker and 20 kV for the Shimadzu. Exact mass (EM) data were recorded on an Agilent Technologies 6230 TOF LC/MS spectrometer. The sample ionization was carried out with a capillary voltage of 3500 V and the electrospray ionization (ESI) conditions included a gas temperature of 325 °C, drying gas flow rate of 8 L/min, and pressure set at 35 psi. MS/MS analysis of the extracted CSP was conducted at the Mick Hitchcock Nevada Proteomics Center at UNR using a ThermoFisher Orbitrap Eclipse Tribrid Mass Spectrometer.

Solid Phase Peptide Synthesis:

All peptide analogs were synthesized following standard Fmoc-based solid-phase peptide synthesis (SPPS) methods with either a peptide synthesizer (CEM Liberty Prime or CEM Liberty1) or via manual synthesis. Peptides were then purified via RP-HPLC to ≥95% purity and their identity verified by high-resolution mass spectrometry. See the Supporting Information for full details.

Circular Dichroism (CD) Spectroscopy:

CD spectra were recorded using a Jasco CD Spectrophotometer (model J-1500–150). See Supporting Information for full experimental details.

Development of S. constellatus reporter strain:

The luminescence-based S. constellatus reporter strain was constructed using previously described protocols with some modifications.²⁸ See Supporting Information for full details.

S. constellatus ΔcomC strain construction:

The S. constellatus ΔcomC background was achieved using homologous recombination to replace the original promoter-comCDE locus with a reversed orientation promoter-comDE sequence and a downstream erythromycin resistance marker, via a linear knockout cassette with 500-bp flanking regions of homology upstream and downstream of the chromosomal insertion site. The ΔcomC cassette was assembled from PCR fragments amplified with primers A1 5, A1 3, Sf 5, Sf 3, Lf 5, Lf 3, A9 5, A9 3, B1 5, B1 3 (Table S2). The transformation of S. constellatus ATCC 27823 (either wild-type or in the PcomX::luc reporter strain) with the linear DNA cassette was achieved via CSP-induced transformation. Briefly, an overnight culture of S. constellatus was diluted 1:200 with fresh THY. An aliquot of 500 μL of diluted culture was transferred to a sterile glass tube and mixed with S. constellatus CSP (113 nM) and 100 ng of the ΔcomC cassette. After incubation at 37 °C with 5% CO₂ for another 3 hours, the culture was streaked on THY agar plates supplemented with 10 μg/mL erythromycin (plus 200 μg/mL spectinomycin for the PcomX::luc reporter strain). Colony formation was assessed after two days. The correct clone was identified and verified with PCR and Sanger sequencing of the entire knockout region to ensure comC was knockout correctly.

Luminescence Bioassays:

Activation Assays:

The ability of synthetic CSP analogs to activate the competence regulon was evaluated using the S. constellatus ATCC 27823 ΔcomC PcomX::luc reporter strain. A single colony of the reporter strain was first cultured at 37 °C with 5% CO₂ in 10 mL of THY media (pH 7.3) containing 200 μg/mL spectinomycin for 16 hours. Following incubation, the bacteria were diluted 1:10 in fresh THY media and incubated at 37 °C with 5% CO₂ until the OD₆₀₀ reached 0.2. During the incubation, clear-bottom white 96-well microtiter plates were prepared for the activation assays. Activation screening was first conducted using a high CSP concentration (10,000 nM) for all CSP analogs. For each experimental sample, 2 μL of a 1 mM CSP stock solution in DMSO was added in triplicate to the wells of the microtiter plate. As a negative control, 2 μL of DMSO was added in triplicate, while 2 μL of a 1 mM stock of the native CSP in DMSO was added in triplicate as a positive control. In the dark, 2 μL of a 15 mg/mL D-luciferin stock solution in distilled water was added to each well. After the addition of CSP and D-luciferin, 196 μL of the diluted bacterial culture was introduced to each well, and the plate was incubated for 30 minutes at 37 °C. Following incubation, both OD₆₀₀ and luminescence were measured for each experimental well. The results were reported as percent activation, calculated by comparing the OD₆₀₀-corrected luminescence of experimental samples to the positive control. Analogs exhibiting over 75% activity relative to the positive control (see Figures S1 and S2) were selected for further evaluation in a dose-dependent assay. In this assay, peptide stock solutions were serially diluted in DMSO (1:2, 1:3, or 1:5) and tested as described above. EC₅₀ values, representing the peptide concentration needed to achieve half-maximal response, were determined using GraphPad Prism. The experiments were performed in triplicate on three separate days.

Inhibition essays:

The ability of synthetic S. constellatus CSP analogs to inhibit S. constellatus ATCC 27823 ΔcomC PcomX::luc reporter strain expression by competing with the native S. constellatus CSP for receptor binding was assessed under the same conditions used for the activation assays. However, during the inhibition screening, the native S. constellatus CSP was added to each well at a fixed concentration (20 nM), which was selected to ensure full activation of the competence regulon, based on dose-response curves generated for the native S. constellatus CSP.

For the positive control, 2 μL of S. constellatus CSP and 2 μL of DMSO were added to each well in triplicate. For the negative control, 4 μL of DMSO was added in triplicate. Next, 2 μL of a 15 mg/mL D-luciferin solution in distilled water was added to each well, either experimental or control. Following this, 194 μL of bacterial culture was added to each well, and the plate was incubated for 30 minutes at 37 °C. After incubation, the OD₆₀₀ and luminescence of each well were measured.

Results were reported as percent activation, which is the ratio between the luminescence (presented as relative luminescence units, RLU/OD₆₀₀) of the analog and that of the positive control (native CSP). Analogs that exhibited significant competitive inhibition in the initial screening (see Figure S3) were further assessed using a dose- dependent assay where peptide stock solutions were serially diluted with DMSO (1:2, 1:3, or 1:5) and tested as described previously. IC₅₀ values for the inhibitor, which represent the concentration needed to reduce QS activation by 50%, were calculated using GraphPad Prism. All experiments were conducted in triplicate over three separate days.

Transformation Assay:

A single colony of S. constellatus ATCC 27823 (wild type or knockout) was cultured in 5 mL of THY media (pH 7.3) at 37 °C with 5% CO₂ for 16 hours. Following incubation, the cultures were diluted 1:20 into fresh THY media. Once the OD₆₀₀ reached approximately 0.2, CSP and other analogs were introduced at specific concentrations. In the wild-type strain, CSP was added at final concentrations of 10,000 nM, 1,000 nM, 100 nM, and 20 nM, along with separate assays using the lead activator CSP I4A (0.5 nM) and lead inhibitor CSP D1A (700 nM). For the knockout strain, CSP (20 nM), CSP I4A (0.5 nM), and CSP D1A (700 nM) were tested. All experimental conditions included 200 ng of pDL278 (Specᴿ). Control assays were conducted either without CSP or without both CSP and the plasmid to assess indigenous competence and antibiotic resistance. After 5 hours of static incubation at 37 °C with 5% CO₂, 20 μL of culture was plated onto THY agar supplemented with 200 μg/mL spectinomycin and incubated for 48 hours to detect positive transformants. This assay was repeated three times in triplicate (triplicate of triplicate).

Biofilm Formation Assay:

Biofilm quantification was assessed using the well-established crystal violet assay with some modifications.^{34,42,43,44,45,46} A single colony of S. constellatus ATCC 27823 (wild type and knockout) was cultured in 5 mL of THY media (pH 7.3) at 37 °C with 5% CO₂ for 16 hours. Following incubation, cultures were diluted 1:100 into fresh THY media supplemented with 1% D-glucose, and 198 μL of the diluted culture was added in triplicate to wells of a 96-well microtiter plate. Each well received either 2 μL of a peptide solution in DMSO at the designated concentration, or DMSO alone as a negative control. Wells containing only THY media with 1% D-glucose and DMSO (without bacteria) were included for background correction. The plate was incubated statically at 37 °C with 5% CO₂ for 24 hours, after which the absorbance at 600 nm (A₆₀₀) was recorded. After incubation, the liquid contents of the wells were gently removed by shaking the plate over a glass basin. Biofilms were washed three times with 250 μL of 1× PBS, then heat-fixed by incubating the plate at 55 °C for 3 hours. Following heat fixation, 200 μL of a 0.1% crystal violet solution was added to each well and left at room temperature for 5 minutes. Excess stains were carefully removed, and the wells were washed twice with 200 μL of water. To solubilize the stained biofilms, 200 μL of 30% (v/v) acetic acid in water was added, and the plate was shaken at 37 °C for 15 minutes. The contents of each well were then diluted 1:5 in water, and absorbance at 595 nm (A₅₉₅) was measured. Each A₅₉₅ reading was normalized to the corresponding A₆₀₀ value. All experiments were performed in triplicate across three independent days. Biofilm formation data are presented as a percentage relative to the untreated wild-type/knockout control, with results expressed as the mean +/− standard deviation of three independent trials.

Isolation of Crude Peptides from Bacterial Supernatants:

Isolation of the native CSP from bacterial supernatants was accomplished using a previously described protocol with some modifications.^16,47 A single colony of S. constellatus ATCC 27823 wild-type strain was grown overnight in 5 mL of THY medium (pH 7.3) at 37 °C under 5% CO₂. The culture was then diluted 1:20 with fresh THY medium to a total volume of 200 mL. Once the OD₆₀₀ reached approximately 0.2, CSP K16A was added, and the culture was incubated for one additional hour. Afterward, the culture was centrifuged at 4,500 rpm for 15 minutes, and the supernatant was filtered through a sterile polyethersulfone (PES) filter and collected in a flask. Ammonium sulfate was added to reach a final concentration of 55% (wt/vol), and the mixture was gently inverted until it dissolved completely. The solution was stored at 4 °C for 24 hours, followed by a second centrifugation at 4,500 rpm for 15 minutes. The supernatant was carefully removed, and the remaining pellet was resuspended in 10 mL of a 1:1 ddH₂O:ACN solution, freeze-dried, and purified via RP-HPLC. The mass of the purified extracted CSP was determined using high-resolution ESI-TOF MS and MS/MS analysis (see Figure S14).

Tandem Mass Spectrometry:

The Streptococcus constellatus CSP solution was prepared in 50:50 methanol:H₂O + 1% formic acid (mass spectrometry grade) to a final concentration of ~0.5 mg/mL. For MS and MS/MS analysis of the extracted CSP peptide, a ThermoFisher Orbitrap Eclipse Tribrid Mass Spectrometer was used. The strongest-intensity precursor ions (MH⁺³ and MH⁺⁴) from the MS data were isolated via quadrupole isolation and subjected to fragmentation (MS/MS) via HCD. During MS/MS analysis, the Orbitrap detector was used with the resolution set to 120,000. The HCD energy was scanned from 0–40% (data not shown) at intervals of 5% to determine the HCD energy range for optimal peptide fragmentation. The HCD energy was set to 36% and 32% for fragmentation of the MH⁺³ and MH⁺⁴ precursor ions, respectively. The spectral data for MS and MS/MS were copied to Microsoft Excel and analyzed in Origin Pro.

Supplementary Material

Supporting Information

The Supporting Information is available free of charge at

Additional experimental procedures, peptide characterization, primary reporter assay data, dose−response curves for CSP analogs, CD spectra of synthetic CSP analogs, and supplementary figures.

Data Availability

All data are presented in the article or ESI.