Structure Activity Relationship Study of the XIP Quorum Sensing Pheromone in Streptococcus mutans Reveal Inhibitors of the Competence Regulon

Abstract

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX-inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly whereas, XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX, an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure-activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Lastly, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

Graphical Abstract

INTRODUCTION

Genetic competence, a horizontal gene transfer mechanism, exists in many bacteria.^1–2 This transient physiological state that allows bacteria to internalize exogenous DNA fragments and integrate them into the genome provides high genetic plasticity.¹ Competence can be induced by various environmental stimuli and has been shown to play a critical role in the acquisition of new phenotypes such as antibiotic resistance and different virulence traits. In many streptococci, competence is regulated by a cell to cell communication system based on secreted peptide signaling molecules.³ This communication mechanism, termed quorum sensing (QS), enables bacteria to assess the population density and coordinate group behavior at high cell number.⁴ Targeting competence regulation in streptococci has received considerable attention and extensive ongoing research efforts are focused on modulating the streptococcus competence regulon as an anti-infective strategy.^5–11

In Streptococcus mutans, the competence regulon plays an important role in its survival and pathogenicity.¹² S. mutans improves its fitness in the oral microflora by increasing genome plasticity through genetic transformation. Moreover, virulence traits such as bacteriocin production and biofilm formation are controlled by the competence regulon.^13–15 At the center of the S. mutans competence regulon is comX, also known as sigX, which encodes an alternative sigma factor that orchestrates a core response by the induction of approximately 30 late competence genes that encode effector proteins necessary for DNA uptake, binding and recombination.^{3, 16}

In S. mutans, comX expression can be induced by two linear peptide pheromones, the CSP (competence stimulating peptide) and the XIP (sigX inducing peptide).¹⁷ These two peptides, CSP and XIP, are dedicated for two individual but interlinked systems, comCDE and comRS, respectively.^{18, 19} CSP induces a bimodal comX expression in complex media, whereas XIP induces a unimodal comX expression in peptide-free chemically defined media (CDM).²⁰ Interestingly, CSP is unable to trigger competence in CDM.^{13, 21} In the comCDE system, CSP, a 21-residue peptide (21-CSP) is derived by cleavage from a propeptide, ComC, and exported through the ATP-binding cassette transporter, ComAB. 21-CSP is further processed by a membrane protease, SepM, into active 18-residue CSP (18-CSP).²² Extracellular 18-CSP then binds to the cognate transmembrane histidine kinase receptor, ComD, leading to phosphorylation of cytoplasmic response regulator, ComE, which functions as an activator of bacteriocin production and immune genes (Figure 1).^{23, 24} ComE also modulates the comRS system through an unknown mechanism and thus induces competence indirectly.^{25, 26}

Figure 1:

Model of CSP and XIP pheromones signaling pathways in S. mutans. In rich media, 21-CSP is processed by SepM into active 18-CSP that binds to the ComD receptor and eventually activates ComE. Activated ComE then initiates the transcription of bacteriocin production-related genes and indirectly the induction of competence (represented by dotted arrow in the figure). In peptide-free medium, ComS, the precursor of XIP, is exported and processed outside the cell to the mature XIP form by an unknown mechanism (represented by dotted arrow in the figure). When XIP reaches a certain threshold concentration, it is transported back into the cell through the oligopeptide permease and forms a complex with the cytosolic receptor ComR. The XIP::ComR complex then upregulates the expression of the comX gene, thus inducing competence directly.

The comRS regulon centrally mediates competence activation in S. mutans. The comRS system includes a cytosolic Rgg-like transcriptional regulator, ComR, and a 17-amino acid peptide precursor, ComS.^{27, 28} In peptide-free medium, ComS is exported by unknown mechanisms, likely a dedicated ABC transporter system, PptAB²⁹ or through cell lysis³⁰ and processed into its mature form, the 7-amino acid XIP (from the C-terminus of ComS).^{27, 28, 31} XIP is transported back into the cell by the oligopeptide permease, Opp, and forms XIP::ComR complex that activates the transcription of comX and comS (Figure 1).^{27, 32}

Although interaction between XIP and ComR is critical for competence induction in S. mutans, detailed structural information about the XIP::ComR interaction is not known yet. In our previous studies, we conducted detailed structure activity relationship (SAR) studies of the CSP pheromone in rich media and identified the critical motifs for both SepM and ComD interactions.^{33, 34} In this study, we set out to perform extensive SAR studies of the XIP pheromone in CDM to identify the structural requirements for effective XIP::ComR interaction. Our results revealed important structural features of XIP that are required for proper ComR binding and activation. Based on the SAR insights we developed multiple inhibitors of the comRS QS system.

RESULTS AND DISCUSSION

We have synthesized the different XIP analogs through standard Solid-Phase peptide synthesis (SPPS) protocols (see the Supporting information for details). All the peptides were purified (>95%) using reversed-phase (RP)-HPLC and their identities were confirmed using high resolution mass spectrometry (HR-MS). All the purified XIP analogs were tested for their ability to modulate comX expression using a S. mutans reporter strain, SMCOM2 (ΔcomC, PcomX::lacZ),³⁵ in CDM (see Materials and Methods for details).

Alanine and d-amino acid scan.

We started our systematic analysis by performing full alanine and d-amino acid scanning of XIP. The alanine scanning revealed several interesting activity trends: First, alanine modification of Gly1, Asp3 or Ser6 was tolerated, resulting in analogs with similar activities to the native XIP (Table 1). Contrary, alanine modification at Leu2 or Trp5 resulted in relatively inactive analogs, highlighting the importance of these side chain residues for effective ComR receptor binding. Interestingly, XIP-W4A (P5) and XIP-L7A (P8) were both able to fully inhibit ComR activation (~90% and ~80% inhibition, respectively) with XIP-W4A (P5) exhibiting moderate inhibitory activity (IC₅₀ = 31 μM) while XIP-L7A (P8) exhibiting weak inhibitory activity (Table 1 and Figure S-3), suggesting that these side chain residues play a role in receptor activation but not receptor binding. The d-amino acid modifications in XIP resulted in completely inactive analogs (Figure S-1 and Table 1), with the exception of XIP-d3 (P10) that was still somewhat active (~45% activation at 100 μM concentration), suggesting that configurational changes of any of the side chains disrupt the proper XIP::ComR interactions.

Table 1.

EC₅₀ or IC₅₀ values of the alanine and d-amino acid scan XIP analogs against the ComR receptor^a

Name	Sequence	EC50 or IC50 (μM)b	95% CIc	Fold changed
XIP (P1)	GLDWWSL	0.44	0.37 – 0.52
XIP-G1A (P2)	ALDWWSL	0.67	0.58 – 0.77	1.5
XIP-L2A (P3)	GADWWSL	--e	--
XIP-D3A (P4)	GLAWWSL	1.0	0.62 – 1.6	2.3
XIP-W4A (P5)	GLDAWSL	31	13 – 73
XIP-W5A (P6)	GLDWASL	--e	--
XIP-S6A (P7)	GLDWWAL	0.26	0.97 – 0.68	0.59
XIP-L7A (P8)	GLDWWSA	> 50
XIP-l2 (P9)	GlDWWSL	--e	--
XIP-d3 (P10)	GLdWWSL	--e	--
XIP-w4 (P11)	GLDwWSL	--e	--
XIP-w5 (P12)	GLDWwSL	--e	--
XIP-s6 (P13)	GLDWWsL	--e	--
XIP-l7 (P14)	GLDWWSl	--e	--

^aSee experimental section for details on reporter strain and methods. See supporting information for primary screening assay results and plots of agonism and antagonism dose response curves. All assays were performed in triplicate.

^bEC₅₀ or IC₅₀ values determined by testing peptides over a range of concentrations.

^c95% confidence interval.

^dRatio where each analog’s EC₅₀ is divided by native XIP EC₅₀; a value <1 indicates a better activator than the parent XIP.

^eEC₅₀ not determined due to the analog’s low induction in primary agonism screening assay.

Conservative point mutations in XIP.

Alanine scan results revealed that the Leu2 and Trp5 residues are critical for receptor binding while the Trp4 and Leu7 residues are important for receptor activation. Therefore, we decided to incorporate conservative point mutations at these four positions using proteogenic and non-proteogenic amino acid residues to further investigate the interactions between these side chains and the binding pocket in ComR. We replaced the aliphatic hydrophobic residues (Leu2 and Leu7) with other aliphatic hydrophobic residues: Ile, Val, norleucine (norLeu), and norvaline (norVal), whereas we replaced the aromatic residues (Trp4 and Trp5) with other aromatic residues: Phe and Tyr, and also with the non-aromatic cyclic side chain residue, beta-cyclohexyl alanine (Cha) (Figure 2).

Figure 2:

Conservative point mutations of Leu and Trp performed in this study.

Starting with Leu2, the loss of chain branching, either while retaining the carbon chain length (Leu → norVal) or coupled with chain elongation (Leu → norLeu) was relatively tolerated, resulting in analogs with activities similar to XIP (less than 5-fold reduction in activity; Table 2). Contrary, changing the position of the carbon chain branching (Leu → Ile) was significantly less tolerated, resulting in an analog 21-fold less active than XIP (Table 2), likely due to steric clashes with the ComR receptor binding pocket. Moreover, chain shortening (Leu → Val) was also detrimental, resulting in an analog with reduced activity (~9-fold change), indicating again that changing the position of the carbon chain branching is detrimental for ComR binding.

Table 2.

EC₅₀ or IC₅₀ values of the conservative mutations of XIP against the ComR receptor^a

Name	Sequence	EC50 or IC50 (μM)b	95% CIc	Fold changed
XIP	GLDWWSL	0.44	0.37 – 0.52
XIP-L2I (P15)	GIDWWSL	9.4	8.2 – 11	21
XIP-L2V (P16)	GVDWWSL	3.9	2.8 – 5.4	8.9
XIP-L2NL (P17)	GnLDWWSL	2.0	1.4 – 3.0	4.5
XIP-L2NV (P18)	GnVDWWSL	0.60	0.32 – 1.1	1.4
XIP-W4F (P19)	GLDFWSL	5.2	4.1 – 6.7	12
XIP-W4Y (P20)	GLDYWSL	> 50
XIP-W4Cha (P21)	GLDChaWSL	--e	--
XIP-W5F (P22)	GLDWFSL	22	10 – 47	50
XIP-W5Y (P23)	GLDWYSL	35	23 – 55	80
XIP-W5Cha (P24)	GLDWChaSL	--e	--
XIP-L7I (P25)	GLDWWSI	0.97	0.93 – 1.0	2.2
XIP-L7V (P26)	GLDWWSV	2.4	1.3 – 4.3	5.5
XIP-L7NL (P27)	GLDWWSnL	3.6	2.6 – 5.1	8.2
XIP-L7NV (P28)	GLDWWSnV	3.0	2.6 – 3.5	6.8

^aSee experimental section for details on reporter strain and methods. See supporting information for primary screening assay results and plots of agonism and antagonism dose response curves. All assays were performed in triplicate.

^bEC₅₀ or IC₅₀ values determined by testing peptides over a range of concentrations.

^c95% confidence interval.

^dRatio where each analog’s EC₅₀ is divided by native XIP EC₅₀; a value <1 indicates a better activator than the parent XIP.

^eEC₅₀ not determined due to the analog’s low induction in primary agonism screening assay. NL, norleucine; NV, norvaline; Cha, cyclohexyl alanine.

Moving to Leu7, chain shortening as well as loss or movement of chain branching were all tolerated, resulting in analogs with similar activities to XIP, while chain elongation (Leu → norLeu) was less tolerated (~8 fold change), likely due to steric clashes with the ComR receptor binding pocket (Table 2). These results suggest that for this side chain residue, the ComR receptor binding pocket is more promiscuous, accommodating relatively well the different modifications. Moreover, for this position, side chains containing γ carbons are required for effective interaction with the ComR receptor that result in receptor activation, as alanine modification resulted in conversion of XIP into a weak competitive inhibitor.

Looking at Trp4, replacement to Phe resulted in a moderate activator, XIP-W4F (P19) exhibiting a 12-fold reduction in activity compared to XIP. Replacement to Tyr converted XIP into a weak inhibitor, XIP-W4Y (P20), while replacement with Cha resulted in a completely inactive analog (Table 2). Combined, these results suggest that side chain planarity, aromaticity, or both play an important role in ComR binding, and that specific electronic/polar effects drive receptor activation. Lastly, the Trp5 position exhibited very high specificity for the indole side chain as Trp → Phe, Tyr or Cha modifications resulted in analogs with very low to no activity (Table 2).

Multiple point mutation XIP analogs.

The alanine scan analysis revealed that alanine modifications at Gly1, Asp3 or Ser6 are tolerated and that alanine modification at Trp4 or Leu7 converted XIP into a competitive inhibitor. Moreover, conservative mutation of Trp4 with Tyr resulted in a weak inhibitor. We therefore wanted to test the additive effect of combining multiple modifications with similar activity trends. Thus, we constructed two multiple modification analogs: the first incorporated the two inhibitory point mutations, W4A and L7A, resulting in XIP-W4AL7A (P29), while the second analog incorporated all the tolerated point mutations, G1A, D3A and S6A, resulting in XIP-G1AD3AS6A (P30). As expected, combining W4A and L7A resulted in a more potent inhibitor (IC₅₀ = 15 μM). Interestingly, the multiple tolerated mutation analog, P30, also exhibited potent inhibitory activity (IC₅₀ = 8.9 μM), however, this analog was found to be a partial agonist (~30% comX activation, see dose response curve in the supporting information). To develop better inhibitors, we then used the multiple tolerated mutation analog, XIP-G1AD3AS6A, as a scaffold and incorporated the W4Y, W4A or L7A mutations into that scaffold, resulting in three inhibitors with no agonism activity, XIP-G1AD3AW4YS6A (P31; IC₅₀ = 22 μM), XIP-G1AD3AW4AS6A (P32; IC₅₀ = 10 μM), and XIP-G1AD3AS6AL7A (P33; IC₅₀ = 11 μM). Lastly, incorporation of the W4A and L7A mutations into XIP-G1AD3AS6A resulted in only a weak inhibitor, P34 (IC₅₀ > 50 μM).

Determining the minimal structural requirements for XIP activity.

The multiple mutation analysis revealed that only two to three pharmacophores are required for effective ComR binding, resulting in XIP-based inhibitors. We therefore set out to determine the minimal XIP sequence required for effective ComR binding and activation. To this end, we performed both N- and C-terminus sequential truncation of XIP. Overall, biological evaluation of the truncated XIP analogs revealed that the C-terminus of XIP is more important for activity than the N-terminus, as truncation of residues from the C-terminus resulted in a more significant reduction in activity (Table 4). Starting with the N-terminus truncated XIP analogs, the biological results suggest that Gly1 is dispensable (0.91-fold change). Further removal of Leu2 and Asp3 from the N-terminus resulted in two inhibitory analogs, P36 and P37 (IC₅₀ = 22 μM for P36 and 7 μM for P37). However, the inhibitory activity was reduced when Trp4 was removed (IC₅₀ = 40 μM), while removal of Trp5 resulted in an inactive di-peptide (SL), P39. Together, these results suggest that a minimum of three residues (From the C-terminus) is required for the XIP analogs to inhibit ComR. Moving to the C-terminus, removal of Leu7 resulted in a weak inhibitor, P40. Further removal of Ser6 resulted in a better inhibitor (P41), however, removal of Trp5 (P42) resulted again in reduced inhibitory activity, while removal of Trp4 resulted in an inactive analog (GLD), P43 (Table 4). Evaluating the N- and C-terminus truncation results, analog P37 (WWSL) exhibited the highest inhibitory activity (IC₅₀ = 7 μM). Starting from this analog (P37) as the scaffold, further removal of the Leu residue resulted in P44 (WWS) that could still effectively inhibit ComR, albeit at slightly reduced potency (IC₅₀ = 12 μM). However, further truncations of P44 from either end resulted in inactive di-peptide analogs: (WS) P45 and (WW) P46, suggesting that P44 (WWS) is the minimal scaffold required for effective ComR binding, and thus inhibitory activity.

Table 4.

EC₅₀ or IC₅₀ values of the N- and C-terminus truncated XIP analogs against the ComR receptor^a

Name	Sequence	EC50 / IC50 (μM)b	95% CIc	Fold changed
XIP	GLDWWSL	0.44	0.37 – 0.52
XIP-Des-G1 (P35)	LDWWSL	0.40	0.21 – 0.77	0.91
XIP-Des-G1L2 (P36)	DWWSL	22	11 – 41
XIP-Des-G1L2D3 (P37)	WWSL	7.0	4.4 – 11
XIP-Des-G1L2D3W4 (P38)	WSL	40	28 – 58
XIP-Des-G1L2D3W4W5 (P39)	SL	--e	--
XIP-Des-L7 (P40)	GLDWWS	> 50
XIP-Des-S6L7 (P41)	GLDWW	25	10 – 63
XIP-Des-W5S6L7 (P42)	GLDW	> 50
XIP-Des-W4W5S6L7 (P43)	GLD	--e	--
XIP-Des-G1L2D3L7 (P44)	WWS	12	8.8 – 17
XIP-Des-G1L2D3W4L7 (P45)	WS	--e	--
XIP-Des-G1L2D3S6L7 (P46)	WW	--e	--

^aSee experimental section for details on reporter strain and methods. See supporting information for primary screening assay results and plots of agonism and antagonism dose response curves. All assays were performed in triplicate.

^bEC₅₀ or IC₅₀ values determined by testing peptides over a range of concentrations.

^c95% confidence interval.

^dRatio where each analog’s EC₅₀ is divided by native XIP EC₅₀; a value <1 indicates a better activator than the parent XIP.

^eEC₅₀ not determined due to the analog’s low induction in primary agonism screening assay.

Inhibitory activity of lead XIP-based analog in the presence of both CSP and XIP.

After we identified several XIP-based QS inhibitors, we set out to test the inhibitory activity of one of the lead inhibitors, P32, in the presence of both 18-CSP and XIP in either CDM or rich media (THY). Our goal was to evaluate the inhibitory activity of the XIP-based inhibitor when both the comCDE and comRS pathways are stimulated. Therefore, we performed the inhibition assay against both the comRS reporter system (SMCOM2 (ΔcomC, PcomX::lacZ)) and the comCDE reporter system (SAB249 (PcipB::lacZ)) (see Materials and Methods for experimental details). In CDM, P32 exhibited significant XIP-dependent inhibition activity against both reporter strains in the presence of both CSP and XIP (Figure 3A–B). Interestingly, P32 exhibited both XIP- and CSP-dependent inhibitory activity in THY when tested against SMCOM2 (Figure 3C). Not surprisingly, in the case of SAB249 in THY, as XIP did not exhibit significant activity, no inhibitory activity was observed for P32 (Figure 3D). Together, our results suggest that XIP-based inhibitors can be used to attenuate the comRS pathway and thus competence in S. mutans, in both chemically defined and rich media.

Figure 3:

Inhibitory activity of the lead XIP-based inhibitor P32 in the presence of both 18-CSP and XIP. (A) Activity of P32 in the presence of 18-CSP (15 nM) and XIP (2 μM) against SMCOM2 in CDM. (B) Activity of P32 in the presence of 18-CSP (30 nM) and XIP (2 μM) against SAB249 in CDM. (C) Activity of P32 in the presence of 18-CSP (15 nM) and XIP (2 μM) against SMCOM2 in THY. (D) Activity of P32 in the presence 18-CSP (30 nM) and XIP (2 μM) against SAB249 in THY. The data represent the mean of nine values (triplicate of triplicate). Error bars indicate standard error of the mean. **, P < 0.0001; *, P < 0.05; and ns, not significant, by student t test.

Evaluating the ability of lead XIP-based inhibitor to modulate QS-regulated phenotypes.

In the last part of this study, we set out to evaluate the potential utility of XIP-based inhibitors as modulators of QS-regulated S. mutans pathogenic phenotypes. To this end, we first evaluated the ability of P32 to inhibit competence induction, a phenotype regulated by XIP, through antibiotic resistance transformation assays. A Spectinomycin resistance plasmid (pDL278) was introduced to wildtype S. mutans (UA159) in the presence of XIP, P32, or both, and incubated for 1 h. The bacteria were then plated on THY agar plates containing Spectinomycin and competence induction was evaluated via colony formation. Expectedly, addition of XIP resulted in many transformants (Figure 4, top section). Contrary, addition of P32 did not yield any observed transformants, and so was the addition of XIP together with P32 (Figure 4, right and bottom sections, respectively). Together these results suggest that the lead inhibitor was able to block competence induction by blocking the comRS QS pathway.

Figure 4:

Transformation assay of S. mutans UA159 in the presence of XIP and P32. The ability of UA159 to internalize a Spectinomycin resistance plasmid (pDL278) was evaluated following treatment with XIP, P32, or XIP + P32. Following treatment with XIP, UA159 was able to internalize Spectinomycin resistance, as could be seen by the number of transformants (top section). Contrary, following treatment with either P32 or XIP + P32, UA159 was unable to internalize Spectinomycin resistance, as determined by the lack of apparent transformants (right and bottom sections, respectively).

We then turned to evaluate the effect QS modulation has on biofilm formation. Previously, biofilm formation in S. mutans was linked to the competence regulon and more specifically to the CSP pathway.^{14, 36} We therefore first tested the ability of 18-CSP or XIP to modulate the amount of biofilm formed following 24 hour incubation using the crystal violet assay.^{37, 38} Unexpectedly, no significant change in biofilm formation was observed for cells treated with 18-CSP, XIP, or both, when compared to the untreated control (Figure S-4). In an attempt to further evaluate the role of the competence regulon in S. mutans biofilm formation, we compared the amount of biofilm formed by UA159 (wildtype) and SMCC3 (ΔcomC mutant). Again, no significant change in the amount of biofilm formed was observed between the two strains (Figure S-5). Previously, Li et al. have shown that a ΔcomC mutant forms biofilms with different structure compared to a wildtype strain.¹⁴ Combining our crystal violet biofilm results with these previous observations, it is tempting to speculate that in S. mutans QS modulation leads to changes in the architecture of biofilms but not in the amount of biofilms formed. However, additional experiments are required to test this hypothesis. Since no observable change in the amount of biofilms formed was detected, the ability of P32 to offset this change could not be tested.

Next, we set out to evaluate the ability of P32 to modulate bacteriocin production, a phenotype under the control of 18-CSP, using an interspecies competition assay against S. anginosus. Since our reporter assay results indicated that P32 modulates the comRS pathway within the competence regulon, we did not expect to observe modulation of bacteriocin production by this compound. Indeed, our interspecies inhibition assay results revealed that P32 neither induces bacteriocin production (Figure 5B) nor attenuates the activation of bacteriocin production by 18-CSP (Figure 5C). Therefore, the interspecies inhibition results further validate the specificity of P32 as a modulator of only the comRS pathway within the competence regulon.

Figure 5:

Interspecies inhibition assay between S. mutans and S. anginosus. S. mutans SMCC3 (ΔcomC) was treated with (A) 15 nM 18-CSP, (B) 100 μM P32, (C) 15 nM 18-CSP + 100 μM P32, or (D) DMSO and tested for its ability to inhibit the growth of S. anginosus ATCC 33397.

Lastly, we set out to evaluate the role of QS in lactic acid production, a hallmark of S. mutans pathogenicity that is responsible to dental caries. Previously, it was demonstrated that pH affects competence regulon expression,³⁹ however, a potential relationship between the competence regulon and lactic acid production was not established. We therefore tested whether 18-CSP or XIP modulate lactic acid production using an EnzyChrome lactate assay. Our results indicate that the competence regulon does not play a significant role in lactic acid regulation, as treatment with either 18-CSP, XIP, P32, or 18-CSP + P32 resulted in similar levels of lactic acid production (Figure S-7).

SUMMARY AND CONCLUSIONS

The comRS QS pathway plays a vital role in competence regulation in S. mutans. In-depth understanding of the key XIP::ComR interaction at the molecular level may lead to the development of anti-infective agents against S. mutans infections. In this study, by performing detailed SAR analysis of XIP in CDM, we revealed the critical role the hydrophobic residues (Leu and Trp) are playing in XIP interaction with the ComR receptor. Moreover, conservative point mutations revealed the chain length requirements of aliphatic hydrophobic residues and specificity of the binding pockets towards planarity/aromaticity of the aromatic residues. Importantly, our SAR analysis of XIP revealed the first S. mutans QS inhibitors. Overall, our systematic analysis enabled us to develop multiple inhibitors of the ComRS system with activities at the low micromolar range. Furthermore, through our analysis, we established the minimal structural features that are required for XIP to effectively bind and activate the ComR receptor. Moreover, we were able to demonstrate that one of the lead inhibitors identified in this study, P32, inhibits both XIP- and CSP-dependent QS activation in conditions where XIP exhibits QS modulation. Lastly, through several phenotypic assays, we were able to exhibit the potential utility of XIP-based inhibitors to attenuate S. mutans pathogenic phenotypes and at the same time validate the specificity of these compounds to the comRS pathway within the competence regulon (Figure 6). These new XIP-based inhibitors may be used as starting scaffolds for the development of anti-infective therapeutics against S. mutans infections.

Figure 6:

A hypothetical model exhibiting the XIP-based inhibitor (P32) proposed mode of action. P32 (red circles) added exogenously (red arrow) outcompetes XIP for the binding of ComR. Upon binding to ComR, P32 blocks comS and comX expression, leading to inhibition of competence.

METHODS

Chemical Reagents and Instrumentation.

All chemical reagents and solvents were purchased from Sigma-Aldrich and used without further purification. Amino acids were purchased from Advanced ChemTech. Water (18 MΩ) was purified using a Millipore Analyzer Feed System. Solid-phase resins were purchased from Advanced ChemTech and Chem-Impex International.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Shimadzu system equipped with a CBM-20A communications bus module, two LC-20AT pumps, an SIL-20A auto sampler, an SPD-20A UV/vis detector, a CTO-20A column oven, and an FRC-10A fraction collector. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data were obtained on a Bruker Microflex spectrometer equipped with a 60 Hz nitrogen laser and a reflectron. In positive ion mode, the acceleration voltage on Ion Source 1 was 19.01 kV. Exact mass (EM) data were obtained on an Agilent Technologies 6230 TOF LC/MS spectrometer. The samples were sprayed with a capillary voltage of 3500 V, and the electrospray ionization (ESI) source parameters were as follows: gas temperature of 325 °C at a drying gas flow rate of 8 L/ min at a pressure of 35 psi.

Biological Assays

Biological Reagents and Strain Information.

All standard biological reagents were purchased from Sigma-Aldrich and used according to enclosed instructions. To examine the QS modulating ability of the XIP analogs, β-galactosidase assays were performed using two reporter strains: S. mutans SMCOM2 (ΔcomC, PcomX::lacZ), and SAB249 (PcipB::lacZ).⁴⁰ Phenotypic assays were performed using S. mutans UA159 (Wt, Sp^s Em^s), S. mutans SMCC3 (ΔcomC),³⁵ and S. anginosus ATCC 33397. For the transformation assays, pDL278 (Escherichia coli-Streptococcus shuttle vector containing Sp cassette; Sp^r)⁴¹ was used to evaluate competence induction.

Preparation of Chemically defined media (CDM).

CDM was prepared following the previously described method⁴² with the following modification: 0.1% glucose was used to make the CDM instead of 1% glucose to minimize the effect of carbohydrate source⁴³ on comX activation.

Inhibitor activity assay in the presence of CSP and XIP in CDM.

The same biological assays were used as described in the Supporting Information to investigate the effect the lead XIP-based inhibitor has on 18-CSP-based activation in CDM. These experiments were done using both S. mutans SMCOM2 (ΔcomC, PcomX::lacZ) and SAB249 (PcipB::lacZ) strains. A total of 6 μL (2 μL of 1.5 μM 18-CSP stock solution for SMCOM2 and 3 μM 18-CSP stock solution for SAB249, 2 μL of 200 μM XIP stock solution, and 2 μL of 10 mM lead XIP-based inhibitor, XIP-G1AD3AW4AS6A (P32), stock solution in DMSO) was added in triplicate to a clear 96-well microtiter plate. In case of one component (either 18-CSP or XIP) or two components (18-CSP + XIP, 18-CSP + inhibitor, XIP + inhibitor) 4 μL or 2 μL DMSO, respectively, were added for a total of 6 μL volume of DMSO. For negative control, 6 μL of DMSO was added in triplicate. Then, 194 μL of bacterial culture in CDM was added to each well. The plate was incubated at 37 °C for 1 h, and the OD 600_nm was measured. The procedure for lysis, incubation with ONPG, and all the measurements were as described in the activation assay (see Supporting Information).

Inhibitor activity assay in the presence of CSP and XIP in THY.

The above experiment was repeated in THY using the same procedure with slight modifications. Sample preparations were the same as described above, with the only difference being the use of THY media. Also, in the case of SMCOM2, bacterial incubation time with the peptides was adjusted to 2 h instead of 1 h (as we previously reported³³). Bacterial incubation time for SAB249 with peptide mixture remained 1 h, as in CDM. The procedure for lysis, incubation with ONPG, and all the measurements were as described in the activation assay (see Supporting Information).

Analysis of Activation/Inhibition Data.

Miller units were calculated using the following formula:

$$\mathit{\text{Miller}} \mathit{\text{Unit}}=1000\times \frac{Ab{s}_{420}-\left(1.75\times Ab{s}_{550}\right)}{t\times v\times Ab{s}_{600}}$$

Abs₄₂₀ is the absorbance of o-nitrophenol (ONP). Abs₅₅₀ is the scatter from cell debris, which, when multiplied by 1.75, approximates the scatter observed at 420 nm. t is the duration of incubation with ONPG in minutes, v is volume of lysate in milliliters, and Abs₆₀₀ reflects cell density.

Transformation assay.

Bacteria from freezer stocks were streaked onto a THY agar plate and incubated for 20–22 h in a CO₂ incubator (37 °C with 5% CO₂). A fresh single colony was transferred to 5 mL of BHI broth and the culture was incubated in a CO₂ incubator overnight (15 h). Overnight culture was then diluted 1:25 with BHI, and the resulting solution was incubated in a CO₂ incubator for 1.5 h. After that, cells were centrifuged at 5000 rpm for 10 min and BHI medium was discarded. Then, cells were washed and centrifuged (5 min at 5000 rpm) two times with 5 mL sterile PBS buffer. PBS buffer was discarded, and the cells were resuspended in CDM media and vortexed for 10 sec followed by 30 min static incubation in a CO₂ incubator at 37 °C. At this point the OD 600_nm was approximately 0.12. Bacteria (970 μL) were added to culture tubes containing one of the following: 10 μL of 200 μM XIP + 10 μL DMSO + 10 μL pDL278 (40 μg/mL); or 10 μL of 10 mM P32 + 10 μL DMSO + 10 μL pDL278 (40 μg/mL); or 10 μL of 200 μM XIP + 10 μL of 10 mM P32 + 10 μL pDL278 (40 μg/mL); or 20 μL DMSO + 10 μL pDL278 (40 μg/mL). After 1 h incubation in a CO₂ incubator, 20 μL of bacterial culture were spread using an inoculation loop on THY plates containing 120 μg/mL Spectinomycin. Then the plates were incubated in a CO₂ incubator for 20 h to 22 h and colony formation was evaluated. This experiment was performed in triplicate and on three separate days (triplicate of triplicate).

Crystal Violet Biofilm Assay:

S. mutans UA159 was grown overnight in BHI media at 37 °C in a CO₂ incubator. The overnight culture was then diluted 100-fold in BHI containing 1% D-glucose and 196 μL of UA159 was added in triplicate to the wells of a 96-well plate. Each well contained either 2 μL of CSP (final concentration 15 nM) and 2 μL of DMSO, 2 μL of CSP (final concentration 15 nM) and 2 μL of XIP (final concentration 2 μM), 2 μL of XIP (final concentration 2 μM) and 2 μL of DMSO, or 4 μL of DMSO (as a negative control). The plate was statically incubated at 37 °C for 24 hours. The absorbance at 600 nm (A600) was recorded. The contents of the wells were carefully decanted by shaking over a glass basin in a biosafety cabinet. Experimental wells were gently washed three times with 250 μL phosphate buffered saline (PBS). The plate was then incubated for 2 h at 55 °C to fix the biofilms. 200 μL of 0.1% (wt/vol) crystal violet in water was then added to each well and allowed to stand at room temperature for 5 min. The well contents were carefully decanted. After that the wells were washed twice with 200 μL water. 200 μL of 30% (vol/vol) acetic acid in water was added to the wells. The plate was shaken at 200 rpm at 37 °C for 15 min before the absorbance at 595 nm (A595) was read. Each A595 value was divided by its corresponding A600 value. The experiment was repeated on three separate days.

To compare the biofilm formation between UA159 (wildtype) and SMCC3 (ΔcomC mutant), the same procedure was followed except that no CSP or XIP were used. Instead of 196 μL of bacterial culture, 200 μL of UA159 and SMCC3 bacterial culture was used in 96 well plates. The experiment was repeated on three separate days.

Interspecies Inhibition Assay.

Fresh colonies of S. mutans SMCC3 and S. anginosus ATCC 33397 from THY agar plates were transferred to sterile tubes containing 5 mL of THY broth, and the tubes were incubated in a CO₂ incubator overnight at 37 °C. The overnight culture of SMCC3 was diluted 1:25 with fresh THY, and the resulting solution was incubated in a CO₂ incubator for 2 h until the bacteria reached early exponential phase (OD₆₀₀ ~ 0.2). Then 970 μL of SMCC3 bacterial culture were added to different sterile cultural tubes containing either 10 μL of 1.5 μM 18-CSP + 20 μL of DMSO, 10 μL of 1.5 μM 18-CSP + 10 μL of 200 μM XIP + 10 μL of DMSO, 10 μL of 1.5 μM 18-CSP + 10 μL of 10 mM P32 + 10 μL of DMSO, 10 μL of 1.5 μM 18-CSP + 10 μL of 200 μM XIP + 10 μL of 10 mM P32, 10 μL of 200 μM XIP + 20 μL of DMSO, 10 μL of 10 mM P32 + 20 μL of DMSO, 10 μL of 200 μM XIP + 10 μL of 10 mM P32 + 10 μL of DMSO, or 30 μL DMSO (negative control). The tubes were then incubated in a CO₂ incubator for 3 h at 37 °C. A 250 μL aliquot of S. anginosus ATCC 33397 overnight culture was spread plated onto a THY agar plate, and the plate was allowed to dry for 15 min. After the plate was dry, wells were made in triplicates for each condition using the larger diameter of a sterile 200 μL pipette tip and using the smaller diameter tip, the agar plugs were picked out. A volume of 60 μL of the different SMCC3 cultures was transferred to each well in triplicate and the plate was incubated in a CO₂ incubator for 24 h at 37 °C. Following the 24 h incubation, the plates were inspected for zones of inhibition around the wells (see Figures 5 and S-6). Each experiment was repeated on three separate occasions.

Lactate Assay.

The lactate assay was performed as previously described with minor modifications.³⁹ Overnight culture of UA159 grown in THY was diluted 1:20 in THY supplemented with 1% glucose. Then 980 μL of bacterial culture were added to different sterile culture tubes containing either 20 μL DMSO, 10 μL of 1.5 μM 18-CSP + 10 μL DMSO, 10 μL of 200 μM XIP + 10 μL DMSO, 10 μL of 10 mM P32 + 10 μL DMSO, or 10 μL of 1.5 μM 18-CSP + 10 μL of 10 mM P32, and the bacterial cultures were incubated in a CO₂ incubator for 3 h at 37 °C. After that, the OD₆₀₀ was measured and bacterial cultures were transferred to 15 mL sterile falcon tubes and centrifuged at 5000 rpm for 10 min. The supernatants were filter sterilized with 0.22 μm 30 mm syringe filters and diluted 1:10 in sterile water, and the lactate assay was performed using the EnzyChrome lactate Assay kit (ECLC-100) according to the manufacturer’s instructions (BioAssay Systems). Lactate production results were normalized by optical density. All experiments were performed in triplicates and repeated on three consecutive days.

Table 3.

EC₅₀ or IC₅₀ values of the multiple mutation XIP analogs against the ComR receptor^a

Name	Sequence	EC50 or IC50 (μM)b	95% CIc
XIP	GLDWWSL	0.44	0.37 – 0.52
XIP-W4AL7A (P29)	GLDAWSA	15	12 – 19
XIP-G1AD3AS6A (P30)	ALAWWAL	8.9	5.9 – 13
XIP-G1AD3AW4YS6A (P31)	ALAYWAL	22	14 – 36
XIP-G1AD3AW4AS6A (P32)	ALAAWAL	10	6.6 – 15
XIP-G1AD3AS6AL7A (P33)	ALAWWAA	11	6.8 – 17
XIP-G1AD3AW4AS6AL7A (P34)	ALAAWAA	> 50

^aSee experimental section for details on reporter strain and methods. See supporting information for primary screening assay results and plots of antagonism dose response curves. All assays were performed in triplicate.

^bIC₅₀ values determined by testing peptides over a range of concentrations.

^c95% confidence interval.