Attenuating the Streptococcus pneumoniae Competence Regulon using Urea-Bridged Cyclic Dominant Negative CSP Analogs

Abstract

Streptococcus pneumoniae (pneumococcus) is a prevalent human pathogen that utilizes the competence regulon quorum sensing circuitry to acquire antibiotic resistance and initiate its attack on the human host. Therefore, targeting the competence regulon can be applied as an anti-infective approach with minimal pressure for resistance development. Herein, we report the construction of a library of urea-bridged cyclic dominant-negative competence stimulating peptide (dnCSP) derivatives and their evaluation as competitive inhibitors of the competence regulon. Our results reveal the first pneumococcus dual-action CSPs, inhibiting the group 1 pneumococcus competence regulon, while activating the group 2 pneumococcus competence regulon. Structural analysis indicate that the urea-bridge cyclization stabilizes the bioactive α-helix conformation, while in vivo studies using a mouse model of infection exhibit that the lead dual-action dnCSP, CSP1-E1A-cyc(Dab6Dab10), attenuates group 1 mediated mortality without significantly reducing the bacterial burden. Overall, our results pave the way to developing novel therapeutics against this notorious pathogen.

Graphical Abstract

INTRODUCTION:

The emergence and rapid evolution of antibiotic resistance in pathogenic bacteria pose a constant threat to the global public health. Streptococcus pneumoniae (pneumococcus) is an opportunistic respiratory human pathogen that rapidly acquires antibiotic resistance through a frequent homologous recombination process controlled by the competence regulon, a quorum sensing (QS) circuitry.^1–4 In the past four decades, S. pneumoniae acquired antibiotic resistance through continuous assimilation of genes from non-competent cells, cell milieus, and genetic exchange between co-colonized species such as Streptococcus mitis and Streptococcus pseudopneumoniae.^{1, 5–9} This recombination-mediated genetic diversity confers a significant advantage to S. pneumoniae by enabling rapid evolution of the genetic information and surface antigens, resulting in resistance against antimicrobial agents and pneumococcal conjugate vaccines.^{1, 10–12} The aforementioned pathogenic properties of S. pneumoniae result in a diverse disease profile, including otitis media, acute pneumonia, pneumonia-derived sepsis, meningitis, and bacteremia in both children and immune-compromised patients.¹³ According to a recent study, S. pneumoniae is the leading cause of lower respiratory tract infections globally, with over 1 million deaths per year.¹⁴

In S. pneumoniae, the competence regulon QS circuit is comprised of five components, ComA-E, and is regulated by an autoinducing peptide termed competence stimulating peptide, CSP (Figure 1).^15–16 The precursor for CSP, ComC, is processed and exported to the extracellular matrix as the mature CSP by the ComAB transporter.^17–18 Upon reaching a threshold concentration, the mature CSP binds and activates its cognate transmembrane histidine kinase receptor, ComD, which in turn, phosphorylates the response regulator, ComE.¹⁹ Activated ComE triggers the transcription of the QS regulatory genes (comABCDE), the alternative sigma factor gene (comX), early competence genes, as well as genes involved in biofilm development and colonization.^20–23 Subsequently, the master regulator ComX activates the expression of late competence genes, including those required in both DNA uptake and transformation, as well as genes involved in virulence factor production.^24–26

Figure 1. Schematic representation of S. pneumoniae competence regulon QS circuit.

The intracellularly expressed pre-CSP encoded from the comC gene gets processed and transported through the ComAB transporter as the mature CSP. Upon reaching a threshold concentration, extracellular CSP binds to and activates its cognate receptor, ComD. Activated ComD phosphorylates the response regulator, ComE, which then upregulates the transcription of the competence regulon genes (comABCDE) as well as comX. The master regulator ComX then upregulates the transcription of genes involved in competence, virulence factor production, and allolysis.

Thus far, two major S. pneumoniae pherotypes, or specificity groups, have been identified, each producing their own signaling molecules, namely CSP1 and CSP2, with their cognate receptors ComD1 and ComD2, respectively.^27–29 Several studies reported that deletion of any component of the competence regulon QS circuitry resulted in attenuation of pathogenicity and consequently in reduction of pneumococcal infections.^{6, 21, 30–34} Indeed, the high therapeutic potential associated with modulating the competence regulon QS circuitry has led to multiple studies focusing on targeting different components of the competence regulon, such as inhibiting CSP export to the extra cellular matrix by disturbing the proton motive force (PMF), degrading the DNA uptake machinery, and inhibiting essential proteins that are responsible for recombination and DNA repair.^35–38

Herein, we set out to design and construct pharmacologically enhanced cyclic peptidomimetic scaffolds capable of attenuating pneumococcal infectivity by targeting the competence regulon QS circuitry through competitive inhibition of the CSP:ComD interaction in both pneumococcal pherotypes. To this end, we chose to utilize side-chain to side-chain peptide cyclization using a urea bridge chemistry. Our goal was to construct cyclic peptides that will: 1) maintain the biologically active conformation of the native signals, CSP1 and CSP2, for optimal ComD1 and ComD2 receptor binding; 2) improve the bioavailability of the peptides by escaping enzymatic degradation; and 3) fine-tune the bioactive conformation to achieve high potency and selectivity by screening the conformational space of the cyclic scaffold through systematic alternation of the macrocycle ring size. Indeed, in this study we report the successful design and synthesis of selective urea-bridged cyclic peptide analogs capable of inhibiting the ComD1 receptor while acting as partial agonists of the ComD2 receptor. Furthermore, pharmacological evaluation of a lead dual-action analog using a mouse model revealed that this peptide has a much longer half-life than the linear native CSP, while remaining non-toxic. Moreover, this dual-action analog is capable of attenuating group 1 pneumococcal infections, but not group 2 infections, further validating its selectivity as well as its potential as a therapeutic agent that does not expose the bacteria to selective pressure for resistance development.

RESULTS AND DISCUSSION:

Design & Synthesis of Urea-Bridged Cyclic dnCSPs:

Our previous studies of the competence regulon and the CSP signals in S. pneumoniae revealed that the bioactive conformation of both CSP1 and CSP2 is an α-helix conformation.^{29, 39} Moreover, our results indicate that the N-terminal domain in both CSPs is vital for activating the ComD1 and ComD2 receptors, respectively, and that Glu1 plays a crucial part in receptor activation.^{29, 40} In CSP1, changing Glu1 to Ala (CSP1-E1A) is sufficient to convert the native signal into a competitive inhibitor, capable of shutting down the competence regulon in S. pneumoniae D39 and attenuating its downstream regulating mechanisms both in vitro and in vivo.^{32, 41} Our structural analysis revealed that within the α-helical central region of both CSP1 and CSP1-E1A, the amino acids (AAs) are very well distributed in two faces (Figure 2A), a receptor binding hydrophobic face (residues L4, F7, F8, F11, and I12) and a modifiable hydrophilic face (residues S5, K6, R9, and D10).³⁹ More recently, Yang et al. revealed that cyclization of CSP1 via a lactam bridge between the AA side chains in positions 6 and 10 could lead to cyclic CSP analogs exhibiting pan-group activities (activating or inhibiting both ComD1 and ComD2) and possessing superior pharmacological properties.⁴²

Figure 2: De novo Peptide Design and Cyclization Strategy:

A) The α-helical backbone structural representation of CSP1-E1A (PDB: 6C00) shows the receptor-binding surface with hydrophobic residues (L4, F7, F8, F11 and I12) highlighted in gray color are aligned on one face of the helix. Hydrophilic residues (S5, K6, R9, D10, and Q14) highlighted in red color are aligned on the opposite face of the helix. The K6 and D10 side chains are in close proximity and were thus used for cyclization (i to i+4). B) Graphical representation of cyclic peptide design with urea-bridge between 6^th and 10^th amino acid side chains (i to i+4). Here, n represents the number of carbon atoms. C) Wide range of macrocycle ring sizes (18–24 atoms) were achieved using various AA side chain lengths of Lys, Orn, Dab, and Dap. D) Workflow of triphosgene-based side-chain to side-chain urea cyclization in a step-wise manner. At first, the linear sequence was synthesized with alloc-protected AAs at positions 6 and 10 for future cyclization. Then, the alloc protective group was selectively removed. Lastly, the free amines were treated with triphosgene to afford a urea bridged macrocycle. See Methods for complete details.

In the current study, we extended our search for lead pan-group inhibitors by changing the cyclization bridge chemistry from a lactam to a urea bond. To this end, we utilized CSP1-E1A as a scaffold and incorporated AAs bearing an amino group-containing side chains at positions 6 and 10 (i→i+4) (Figure 2B and 2C). Following the successful synthesis of the fully protected linear sequence on solid support and selective deprotection of the two side-chain amino groups, on-resin cyclization was carried out using triphosgene (Figure 2D). The urea-bridged cyclic peptides were cleaved from the resin and purified to homogeneity (≥ 95% purity) using semi-preparative HPLC (see details in the Materials and Methods section). To fine-tune the conformation of the cyclic peptides, we designed a systematic library of 16 cyclic peptides bearing various ring sizes and bridge positions on the macrocycle region, and their linear counterparts (Table S1 and S2). We achieved a wide range of macrocycle ring sizes, varying from 18 atoms to 24 atoms, with multiple permutations of Lys and its shorter side chain length versions: Ornithine (Orn), 2,4-diaminobutyric acid (Dab), and 2,3-diaminopropionic acid (Dap) (Figure 2C). Using these non-proteogenic amino acid substitutions and cyclization strategy, we aimed to develop superior dnCSPs that can escape cellular enzymatic degradation and exhibit high therapeutic potential.

Biological Evaluation of Cyclic dnCSPs:

Our objective was to use CSP1-E1A as a scaffold for the development of dnCSPs that target the competence regulon QS circuit in S. pneumoniae. To this end, we utilized the previously described S. pneumoniae D39 and S. pneumoniae TIGR4 β-galactosidase reporter strains to investigate the structure-activity relationships (SARs) of all 32 CSP1-E1A-based analogs constructed in this study (16 cyclic and 16 linear analogs; see the Materials and Methods section for more details). All the peptides were initially screened at a high peptide concentration (10,000 nM) to identify analogs capable of activating the ComD receptors (Figure S1–S4). Analogs that exhibited >50% activation compared to the native signal (CSP1 or CSP2, respectively) were further evaluated (Figure S5–S8) and their EC₅₀ values were determined (Table 1–2 and Table S5). Analogs that failed to activate the ComD receptors, <50% activation compared to the native signal, were assessed for their ability to act as competitive inhibitors. Analogs that exhibited >50% inhibition of the native signal were further evaluated and their IC₅₀ values were determined (see the supporting information for dose response curves).

Table 1:

Biological activity and structural properties of CSP1-E1A cyclic analogs^[a]

	Peptide Name	Ring Size (Atoms)	IC50 (nM)[b] (95% CI)[c]	EC50 (nM)[b] (95% CI)[c]	% Helicity in 20% TFE (PBS)
			ComD1	ComD2
Linear Analogs[d]	CSP1	---[f]	---	526 (498–556)	20.1 (1.0)
	CSP2	---[f]	---	50.7 (40.6–63.2)	β-sheet
	CSP1-E1A	---[f]	85.7 (50.7–145)	---[g]	18.8 (1.0)
	CSP1-E1A-cyc(K6K10)	24	298 (157–574)	---[g]	21.6 (9.0)
	CSP1-E1A-cyc(K610Orn)	23	264 (159–438)	---[g]	18.6 (5.8)
	CSP1-E1A-cyc(Orn6K10)	23	81.3 (47.7–138)	---[g]	19.6 (6.6)
	CSP1-E1A-cyc(Dab6K10)	22	395 (328–476)	449 (376–537)	30.3 (10.6)
	CSP1-E1A-cyc(K6Dab10)	22	811 (748–880)	274 (226–332)	8.9 (2.1)
	CSP1-E1A-cyc(Orn6Orn10)	22	>1000	>1000	5.8 (3.5)
	CSP1-E1A-cyc(K6Dap10)	21	448 (306–655)	240 (205–282)	31.1 (8.3)
	CSP1-E1A-cyc(Dap6K10)	21	509 (307–845)	---[g]	28.9 (14.3)
	CSP1-E1A-cyc(Orn6Dab10)	21	>1000	73.2 (47.8–112)	16.1 (3.9)
	CSP1-E1A-cyc(Dab6Orn10)	21	>1000	---[g]	29.7 (5.9)
	CSP1-E1A-cyc(Orn6Dap10)	20	>1000	224 (132–378)	14.1 (6.4)
	CSP1-E1A-cyc(Dap6Orn10)	20	252 (150–423)	269 (152–475)	19.6 (7.8)
	CSP1-E1A-cyc(Dab6Dab10)	20	89.8 (42.7–189)	56.3 (34.3–96.3)	20.6 (3.5)
	CSP1-E1A-cyc(Dap6Dab10)	19	176 (111–279)	4.76 (2.59–8.76)	18.7 (0.8)
	CSP1-E1A-cyc(Dab6Dap10)	19	129 (102–161)	>1000	7.8 (0.7)
	CSP1-E1A-cyc(Dap6Dap10)	18	199 (128–307)	>1000	12.4 (0.3)

^[a]See the Supporting Information for agonism and antagonism dose-response curves and CD data. All SAR assay data were averaged over three independent experiments.

^[b]IC₅₀ and EC₅₀ values were calculated by testing peptides over a range of concentrations.

^[c]95% confidence interval.

^[d]Previously reported value from Ref²⁹.

^[e]Peptide from current study.

^[f]Ring size is not applicable for linear sequences.

^[g]EC₅₀ not determined due to the analog’s low induction in the primary agonism screening assay.

Table 2:

Biological activity and structural properties of CSP1-E1A linear analogs^[a]

	Peptide Name	IC50 (nM)[b] (95% CI)[c]		EC50 (nM)[b] (95% CI)[c]	% Helicity in 20% TFE (PBS)
		ComD1	ComD2	ComD2
Linear Analogs[d]	CSP1	---	---	526 (498–556)	20.1 (1.0)
	CSP2	---	---	50.7 (40.6–63.2)	β-sheet
	CSP1-E1A	85.7 (50.7–145)	---[f]	---[g]	18.8 (1.0)
	CSP1-E1 AK6DapD10E	>1000	769 (571–1035)	---[g]	---
	CSP1-E1AD10K	217 (116–405)	---[f]	---[g]	21.5 (1.0)
	CSP1-E1AD10Orn	282 (172–462)	---[f]	---[g]	16.0 (0.3)
	CSP1-E1AD10Dab	236 (136–410)	---[f]	---[g]	14.4 (1.0)
	CSP1-E1AD10Dap	56.1 (35.3–89.1)	---[f]	---[g]	25.1 (2.2)
	CSP1-E1AK6OrnD10K	339 (217–538)	>1000	---[g]	17.7 (0.4)
	CSP1-E1AK6OrnD10Orn	641 (431–954)	>1000	---[g]	13.5 (0.9)
	CSP1-E1 AK6OrnD 10Dab	148 (104–211)	>1000	---[g]	22.6 (0.8)
	CSP1-E1 AK6OrnD 10Dap	24.7 (14.0–43.5)	>1000	---[g]	30.1 (2.5)
	CSP1-E1 AK6DabD10K	784 (681–902)	>1000	---[g]	13.8 (1.1)
	CSP1-E1 AK6DabD10Orn	405 (273–601)	>1000	---[g]	16.0 (1.0)
	CSP1-E1 AK6DabD10Dab	428 (257–707)	>1000	---[g]	18.4 (1.1)
	CSP1-E1 AK6DabD10Dap	118 (85.1–165)	>1000	---[g]	23.8 (1.5)
	CSP1-E1 AK6DapD10K	454 (359–575)	---[f]	39.5 (27.7–55.9)	22.8 (1.8)
	CSP1-E1 AK6DapD10Orn	386 (363–411)	---[f]	82.3 (48.8–138)	16.0 (1.0)
	CSP1-E1 AK6DapD10Dab	145 (88.7–237)	---[f]	44.5 (25.1–78.8)	34.3 (0.4)
	CSP1-E1 AK6DapD10Dap	12.4 (8.85–17.4)	---[f]	22.1 (16.1–30.5)	29.5 (1.1)

^[a]See the Supporting Information for agonism and antagonism dose-response curves and CD data. All SAR assay data were averaged over three independent experiments.

^[b]IC₅₀ and EC₅₀ values were calculated by testing peptides over a range of concentrations.

^[c]95% confidence interval.

^[d]Previously reported value from Ref²⁹.

^[e]Peptide from current study.

^[f]IC₅₀ not determined due to the analog’s low induction in the primary antagonism screening assay.

^[g]EC₅₀ not determined due to the analog’s low induction in the primary agonism screening assay.

The initial screening revealed that all the cyclic analogs inhibit the ComD1 receptor (Figure S1). Surprisingly, most of the cyclic analogs (11 out of 16) were found to activate the ComD2 receptor (Figure S3). These results are in striking contrast to previously published data, all of which supporting the notion that the N-terminal region of CSP1 and CSP2 is solely responsible for ComD receptor activation, and that the E1A modification is sufficient to abolish the CSP activation ability, resulting in either competitive inhibitors or completely inactive analogs.^{29, 40, 43–44} However, the results reported herein suggest that modifications in the central region of the CSP scaffold could lead to activating analogs, either via a conformational change that support productive interaction with the ComD receptor, or by directly compensating for the lost interactions between the modified N-terminal region and the ComD receptor (see detailed analysis below).

In the study by Yang et al. the authors revealed that a macrocycle ring size of 18 to 19 atoms is optimal for a 6–10 (i→i+4) lactam cyclization.⁴² Moreover, the identity of the AA residues in positions 6 and 10 were found to be critical for ComD2 modulation,²⁹ as cyclic peptides bearing an Asp residue at position 10 were unable to modulate ComD2, whereas cyclic peptides bearing a Glu residue in position 10 and short amino-containing side chain in position 6 (Dab or Dap) were found to effectively modulate ComD2 activity.⁴² Consequently, the authors identified CSP1-E1A-cyc(Dap6E10) as a highly potent pan-group dnCSP, both in vitro and in vivo.⁴² In contrast, biological evaluation of the current urea-bridged cyclic peptides revealed that these peptides could only effectively inhibit the ComD1 receptor but not the ComD2 receptor. Moreover, within this library, macrocycle sizes of 23–24 and 18–20 atoms were found to be optimal for inhibitory activity against ComD1, whereas macrocycle size of 21–22 atoms resulted in weak ComD1 inhibitors (Table 1). The best ComD1 urea-bridged cyclic inhibitor identified was CSP1-E1A-cyc(Orn6K10), with an IC₅₀ value of 81.3 nM (with a ring size of 23 atoms), similar to the parent scaffold CSP1-E1A (IC₅₀ value of 85.7 nM). Two additional urea-bridged cyclic analogs, CSP1-E1A-cyc(Dab6Dab10) (with a ring size of 20 atoms) and CSP1-E1A-cyc(Dab6Dap10) (with a ring size of 19 atoms), were found to be potent ComD1 inhibitors, exhibiting IC₅₀ values of 89.8 and 129 nM, respectively (Table 1).

Biological Evaluation of Linear dnCSPs:

To gain additional insight as to the effects our modifications to the 6^th and 10^th positions of CSP1-E1A had on the peptide activity, we evaluated the pre-cyclic linear counterparts for their ability to modulate QS in both S. pneumoniae specificity groups. All the linear analogs exhibited inhibitory activity against the ComD1 receptor, and out of the 16 analogs, the 8 analogs possessing the permutations K6Orn or K6Dab also exhibited inhibitory activity against the ComD2 receptor. Unfortunately, none of the linear analogs exhibited significant potency against the ComD2 receptor, classifying them as weak pan-group inhibitors (Table 2). The best ComD1 inhibitor observed was CSP1-E1AK6DapD10Dap with an IC₅₀ value of 12.4 nM, 7-fold better than the parent scaffold, CSP1-E1A. Additional potent ComD1 inhibitors identified in this study were CSP1-E1AK6OrnD10Dap (IC₅₀ values of 24.7) and CSP1-E1AD10Dap (IC₅₀ values of 56.1), exhibiting a 3.5-fold and 1.5-fold increased potency compared with the parent scaffold, CSP1-E1A (Table 2 and Figure 3A). Interestingly, all three lead ComD1 inhibitors possess the Dap residue in position 10, suggesting that the replacement of the negatively charged residue Asp with a short positively charged residue (Dap) is highly tolerated and maintains productive interactions with the ComD1 receptor (Figure 3B). Moreover, the activity trend between the three lead analogs suggests that the replacement of Lys at position 6 with a shorter side chain residue (Dap or Orn) is beneficial for ComD1 interaction, leading to highly potent ComD1 inhibitors (Figure 3A).

Figure 3: Optimized binding of ComD:CSP interactions.

A) Bar graph representation of IC₅₀ values for a select CSP1-E1A linear analogs against the ComD1 receptor. CSP1-E1A analogs bearing substitutions with the short, positively charged side chain (Dap) at position 10 exhibited high inhibitory potency against ComD1. We observed a decrease in IC₅₀ value with a decrease in AA side chain length at the 10^th position with the lowest IC₅₀ value being 12.4 nM, CSP1-E1AK6DapD10Dap. B) Graphical representation of optimized binding for activation (upper panel) and inhibition (lower panel). In the natural CSP1, side chains E1 and D10 are involved in activating the ComD1 receptor, and conversely, E1A and D10Dap are involved in inhibiting the ComD1 receptor.

Cyclic and Linear E1A Analogs Activate the ComD2 Receptor:

As mentioned above, previous SAR studies revealed that the N-terminus region of S. pneumoniae CSPs is involved in ComD receptor activation and that the E1A modification is sufficient to convert CSP1 into a competitive ComD1 inhibitor^{29, 40}. Surprisingly, initial agonism screening assays revealed that most of the CSP1-E1A cyclic analogs (11 out of 16) as well as the four linear CSP1-E1A analogs bearing the K6Dap modification can activate the ComD2 receptor. Interestingly, four potent ComD1 inhibitors, two cyclic analogs, CSP1-E1A-cyc(Dab6Dab10) and CSP1-E1A-cyc(Dap6Dab10), and two linear analogs, CSP1-E1A-K6DapD10Dab and CSP1-E1A-K6DapD10Dap, were found to be potent ComD2 activators, although these four analogs could not fully activate the ComD2 receptor (~60–75% activation compared to CSP2), classifying them as partial agonists (Figure 4). Of the two cyclic dual activity analogs, CSP1-E1A-cyc(Dab6Dab10) was found to activate the ComD2 receptor and inhibit the ComD1 receptor in about the same concentration (EC₅₀ value of 56.3 nM against ComD2 and IC₅₀ value of 89.8 nM against ComD1) (Figure 4A, Table 1 and Table S5), making it a valuable tool to evaluate the effect QS modulation has on the selection between the two pneumococcal specificity groups during co-infections.

Figure 4: ComD Activation and Inhibition Dual behavior.

The dose curves, agonism (blue) and antagonism (red), of the two lead molecules, CSP1-E1A-cyc(Dab6Dab10) (A) and CSP1-E1AK6DapD10Dap (B), exhibiting inhibitory activity against ComD1 and partial agonistic activity against ComD2 are presented.

Moving to the linear analogs, all four CSP1-E1A analogs bearing the K6Dap modification, were found to be potent ComD2 activators, but differed in their inhibitory potency against the ComD1 receptor (Table 2). When comparing these results to previous studies, the closest peptide sequence to these analogs to be evaluated was CSP1-E1AK6DapD10E.⁴² Interestingly, this analog was found to be a weak ComD2 inhibitor.⁴² Thus, our results suggest that the replacement of the negatively charged AA (Glu) with positively charged AA at the 10^th position, irrespective of chain length, converts the CSP1-E1AK6Dap scaffold from inhibitory against ComD2 to agonistic against ComD2, while maintaining the inhibitory characteristic against ComD1 (Figure 4B).

Secondary Structure Analysis using Circular Dichroism (CD):

Previously, we and others have reported a strong correlation between α-helicity and bioactivity of CSPs.^{29, 39, 42, 45} Since our current SAR analysis revealed that the macrocycle ring size and bridge position strongly affect the functional behavior of the urea-bridged cyclic CSPs, we evaluated whether the functionality changes are the result of secondary structure alteration. To this end, we recorded far-UV CD (195–260 nm) in both aqueous conditions (PBS, pH 7.4) as well as in membrane mimicking environment (20% TFE in PBS, pH 7.4) at 25 °C for all the cyclic and linear peptide analogs (Figure 5, S9, and S10). In membrane mimicking conditions, all the cyclic and linear analogs exhibited an α-helical conformation with a characteristic negative minima at 208 nm and 222 nm. Furthermore, peptide cyclization was found to stabilize the helical conformation as the cyclic analogs exhibited α-helical characteristics in aqueous conditions, whereas the linear analogs did not (Figure 5). To further evaluate the effects peptide cyclization, ring size, and bridge position had on peptide structure, the percent helicity was calculated for all the peptides exhibiting an α-helical characteristics using the mean residue ellipticity at 222 nm, as previously described.²⁹ Regarding the linear analogs, secondary structure analysis revealed a direct and strong correlation between percent helicity and bioactivity. Analogs that exhibited high helicity also displayed a significant increase in activity, and vice versa (see Figure 5B and Table 2). As for the cyclic peptides, analogs with a macrocycle ring size of 23–24 and 20–19 atoms were observed to be potent QS-modulators. These peptides also exhibited high percent helicity compared to the relatively less active analogs with a macrocycle ring size of 21–22 atoms (see Table 1 and Figure 5A for representative CD spectra of peptides from each macrocycle ring range and their percent helicity). Overall, based on the structural and functional characterization of the cyclic analogs, it is tempting to speculate that the observed activity trend is a result of a conformational change, the shorter macrocycles (18–20 atoms) are locked in a rigid bioactive conformation, the medium length macrocycles (21–22 atoms) are locked in a slightly different, non-active conformation, while the longer macrocycles (23–24 atoms) have sufficient flexibility to adopt the bioactive conformation, resulting in analogs with similar activities to those of the shorter macrocycles. However, in-depth structural characterization is required to evaluate the validity of this hypothesis.

Figure 5: Secondary structure analysis of cyclic (A) and linear (B) analogs using far-UV CD measurements.

All the measurements were performed with a peptide concentration of 100 μM or 200 μM, depending on peptide solubility. We observed the characteristic negative minima at 208 nm and 222 nm in the far-UV CD profile, which corresponds to an α-helix in membrane mimicking milieu (20% TFE in PBS, pH 7.4). In the case of aqueous solution (PBS, pH 7.4), cyclic peptides exhibit an α-helix conformation, whereas linear analogs exhibit a random coil structure. The percentage of helicity acquired in TFE is directly correlated with biological activity for the CSP1-E1A analogs (see Table 1 and 2, and the Supporting Information for complete details). Mean residue ellipticity (MRE) reported in units of deg cm² dmol⁻¹ residue⁻¹.

Pharmacological Evaluation of Lead Cyclic dnCSP

We next evaluated the potential utility of the lead dual-action cyclic dnCSP, CSP1-E1A-cyc(Dab6Dab10), both as a probe to assess the effect of differential QS modulation (activation of one group while inhibition of the other) on the selection between the two pneumococcal specificity groups, as well as a therapeutic agent against group 1 pneumococcal infections. To this end, we first confirmed the activity trend observed in the reporter assays by evaluating the ability of CSP1-E1A-cyc(Dab6Dab10) to inhibit the release of pneumolysin (PLY), a major pneumococcal virulence factor, by conducting an in vitro hemolysis assay. It has been well-established that activation of the competence regulon results in allolysis, where the autolysins (LytA, CbpD, and CibAB) are released, attack, and lyse neighboring non-competent cells, and that PLY is released in the process.^{33, 46} In the hemolysis assay, D39 or TIGR4 were treated with increasing concentration of CSP1-E1A-cyc(Dab6Dab10), in the presence or absence of the native signal, CSP1 or CSP2, respectively. Following incubation, extracellular PLY in the culture supernatant was filter-sterilized and enriched, and added to red blood cells. Expectedly, CSP1-E1A-cyc(Dab6Dab10) was found to inhibit autolytic release of PLY in D39 (Figure 6A), accompanied by diminished hemolytic activity (Figure 6C). In contrast, CSP1-E1A-cyc(Dab6Dab10) amplified the release of PLY in TIGR4 caused by CSP2 (Figure 6B), which significantly increased blood hemolysis (Figure 6D).

Figure 6: CSP1-E1A-cyc(Dab6Dab10) competitively inhibits pneumolysin release and hemolysis induced by CSP1.

(A-D) Group 1 strain D39 and group 2 strain TIGR4 were treated with 45 or 46 nM of CSP1 or CSP2, respectively, in the presence or absence of increasing concentrations of CSP1-E1A-cyc(Dab6Dab10). The release of pneumolysin into culture supernatant (A, B) and the corresponding hemolytic activity (C, D) were quantified. All experiments were performed in triplicate. Data are shown as the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 or ***p < 0.0001 against D39 exposed to CSP1 or TIGR4 exposed to CSP2 as determined by two-way ANOVA with Tukey’s multiple comparisons tests.

We then evaluated the ability of CSP1-E1A-cyc(Dab6Dab10) to attenuate pneumococcal infection in a mouse model of acute pneumonia. Mice were intranasally infected with either D39 or TIGR4, then treated twice at 2- and 24 hours post infection (hpi) with either PBS (vehicle, negative control) or CSP1-E1A-cyc(Dab6Dab10) (4 mg/kg), and mouse mortality was monitored for 120 hpi. For D39-infected mice treated with the vehicle, 100% mortality was observed within 60 hpi. CSP1-E1A-cyc(Dab6Dab10) attenuated both mouse mortality as well as the kinetic of death in D39-infected mice (Figure 7A). In contrast, for mice infected with TIGR4, treatment with either the vehicle or CSP1-E1A-cyc(Dab6Dab10) yielded similar results, with approximately 50% mouse mortality in both groups 72 hpi (Figure 7B). These results further validate the activity of CSP1-E1A-cyc(Dab6Dab10) as a selective group 1 inhibitor.

Figure 7: CSP1-E1A-cyc(Dab6Dab10) attenuates mouse mortality during acute pneumonia by the group 1 D39 strain.

CD-1 mice (n=15 per cohort) were intranasally infected with D39 (4.5×10⁶ CFU/mouse) or TIGR4 (1.07×10⁶ CFU/mouse) and treated twice intravenously through retro-orbital injection with either PBS or CSP1-E1A-cyc(Dab6Dab10) [4 mg/kg] at 2 hpi and 24 hpi. Mouse mortality was monitored for 120 hpi. A) Mortality of mice infected by D39. B) Mortality of mice infected by TIGR4.

Because of the unique ability of CSP1-E1A-cyc(Dab6Dab10) to inhibit competence dependent virulence in group 1 strain D39 while activating the same process in the group 2 strain TIGR4, we examined if the dnCSP could confer advantages to TIGR4 during a competitive infection against D39. Moreover, the serotype 2, D39 strain (ComD1), and serotype 4, TIGR4 strain (ComD2) are the most frequently used strains in pneumococcal pathogenesis studies with their complete genome sequences reported with annotations. We genetically fused the erythromycin and kanamycin resistance genes, respectively, in D39 and TIGR4, downstream of the constitutively expressed rplL gene to allow for the selection and quantification of each strain in the mixed inoculum as well as in the output from lungs and spleens. Mice (10 per cohort) were intranasally inoculated with D39-rplL-Erm^R (2.5×10⁶ CFU/mouse) and TIGR4-rplL-Kan^R (2.5×10⁶ CFU/mouse) in a 1:1 mixture of both pneumococcal strains (total dose of 5×10⁶ CFU/mouse). Infected mice were treated twice (2 and 24 hpi) with either PBS (vehicle control) or CSP1-E1A-cyc(Dab6Dab10) (4 mg/kg), then euthanized at 48 hpi to determine the bacterial burdens in both the lungs and the spleens (Figure 8). In general, the data revealed that TIGR4 was less virulent, yielding a lower bacterial burden in lungs with lesser invasive spread to the spleens compared to D39 (Figure 8A, B). CSP1-E1A-cyc(Dab6Dab10) treatment reduced the D39 burden in coinfection by ~ 0.7 log, but not in the TIGR4, confirming that the dnCSP has divergent activities on group 1 versus group 2 pherotypes. These results suggest that CSP1-E1A-cyc(Dab6Dab10) does not have a direct effect in clearing the bacteria, hence, it does not expose the bacteria to selective pressure for resistance development, but nonetheless, it is capable of producing the desired end-result of attenuated mortality due to reduced pathogenicity.

Figure 8. Competitive infection analysis of D39 and TIGR4 in mice treated with CSP1-E1A-cyc(Dab6Dab10).

CD-1 mice (10 per cohort) were intranasally inoculated with a 1:1 mixture of D39-rplL-Erm^R (2.5×10⁶ CFU/mouse) and TIGR4-rplL-Kan^R (2.5×10⁶ CFU/mouse), with a total dose of 5×10⁶ CFU/mouse. Infected mice were treated twice (2 and 24 hpi) with either PBS (vehicle control) or CSP1-E1A-cyc(Dab6Dab10) (4 mg/kg), then euthanized at 48 hpi to determine the bacterial burdens in both the lungs and the spleens. Homogenized lungs or spleens were plated on THY agar (for total output), THY supplemented with erythromycin (250 ng/mL; select for D39-rplL-Erm^R) or THY supplemented with kanamycin (200 ng/mL; select for TIGR4-rplL-Kan^R). A) Bacterial burdens in the lungs at 48 hpi. B) Bacterial burden in the spleens at 48 hpi. Total D39+TIGR4 output-PBS: total pneumococcal burden that include both D39-rplL-Erm^R and TIGR4-rplL-Kan^R after PBS treatment. Total D39+TIGR4 output-CSP1-E1A-cyc(Dab6Dab10): total pneumococcal burden after treatment with CSP1-E1A-cyc(Dab6Dab10). D39 output-PBS: Burden of D39-rplL-Erm^R from the total D39+TIGR4 output-PBS. D39 output-CSP1-E1A-cyc(Dab6Dab10): Burden of D39-rplL-Erm^R from Total D39+TIGR4 output-CSP1-E1A-cyc(Dab6Dab10). TIGR4 output-PBS: burden of TIGR4-rplL-Kan^R from the total D39+TIGR4 output-PBS. TIGR4 output-CSP1-E1A-cyc(Dab6Dab10): burden of TIGR4-rplL-Kan^R from total D39+TIGR4 output-CSP1-E1A-cyc(Dab6Dab10).

Finally, we wanted to evaluate the in vivo stability of the lead dual-action cyclic dnCSP, CSP1-E1A-cyc(Dab6Dab10), in comparison to the native signal, CSP1 as well as its potential toxicity. For the stability studies, we labeled both peptides with Cyanin7.5 (Cy7.5) and performed IVIS imaging to quantify the degradation and clearance of the peptides over time. Our results indicate that it takes 36 hours to clear intravenously-injected CSP1, whereas CSP1-E1A-cyc(Dab6Dab10) lasts 86 hours before it gets fully cleared (Figure 9). These results highlight the stabilization conferred by peptide cyclization, as the two scaffolds are very similar, with the exception of the presence of the macrocycle. As for the toxicity studies, we examined the in vivo safety profiles of CSP1-E1A-cyc(Dab6Dab10). After 1 week of exposure at therapeutic dose, mice continued to gain weight (Figure S14). Hematological and serum blood chemistry analyses as well as histopathological examination of vital organs indicate that CSP1-E1A-cyc(Dab6Dab10) did not induce abnormal toxicity (Tables S6–S8 and Figures S15–S19). For assay details and additional information, please see the Supporting Information.

Figure 9: CSP1-E1A-cyc(Dab6Dab10) exhibits superior biostability compared to CSP1.

The synthetic peptides were labeled with Cyanin7.5 (Cy7.5), and 50 μg was intravenously injected into CD-1 mice (n=4–5) through the retro-orbital route, and monitored for degradation and clearance by using the IVIS SpectrumCT imaging system. The excitation/emission settings of IVIS were set to 745nm/800nm, and imaging was performed at the indicated time points. A) The fluorescence signal of CSP1-Cy7.5. B) The fluorescent signal of CSP1-E1A-cyc(Dab6Dab10)-Cy7.5.

CONCLUSIONS:

Pneumococcus utilizes the competence regulon QS circuitry to acquire antibiotic resistance genes and initiate its attack on the human host. Blocking the competence regulon QS circuitry can thus be exploited to attenuate pneumococcal infections. Herein, we report the design and synthesis of a library of urea-bridged cyclic dnCSPs capable of blocking the competence regulon QS circuitry in group 1 pneumococcus. Our results indicate that macrocycle ring sizes of 18–20 and 23–24 atoms are optimal for inhibitory activity against the ComD1 receptor. Moreover, our results reveal, for the first time, that analogs bearing the E1A modification, previously reported to be a key modification in the conversion of CSPs into competitive ComD inhibitors, can activate non-cognate ComD receptors, resulting in a few analogs with dual-action activity, that is inhibiting ComD1 while activating ComD2. Lastly, structural analysis using CD spectroscopy exhibited that the urea-bridge cyclization stabilizes an α-helix conformation required for effective ComD receptor binding.

Phenotypic analysis of the lead dual-action cyclic dnCSP, CSP1-E1A-cyc(Dab6Dab10), revealed that this analog is capable of blocking PLY release in group 1 pneumococcus, but not group 2. Furthermore, in a mouse model of infection, CSP1-E1A-cyc(Dab6Dab10) was able to attenuate mouse mortality caused by group 1 pneumococcus, while not significantly affecting bacterial burden, suggesting that the peptide is capable of inhibiting virulence without exposing the bacteria to selective pressure for resistance development. Lastly, pharmacological evaluation of CSP1-E1A-cyc(Dab6Dab10) in comparison to the native signal, CSP1, highlight the therapeutic potential of this analog, as it exhibited enhanced pharmacological properties, being detected by IVIS for up to 86 hours, compared to CSP1, who was cleared from the animal after 36 hours, while remaining non-toxic.

Overall, our results highlight the therapeutic potential of targeting the competence regulon as an anti-virulence approach as well as the prospect of utilizing signal-based competitive inhibitors to block the competence regulon as a novel class of antimicrobial agents that attenuate the infection without exposing the bacteria to selective pressure for the development of resistance. Future studies should focus on improving the cyclic dnCSP scaffold to enhance both its activity and pharmacological properties, as well as conduct more in-depth pharmacological characterization of the lead analogs to solidify their potential as next generation therapeutics. Such studies are ongoing in our labs and will be reported in due course.

MATERIALS AND METHODS:

Reagents and instrumentation:

N,N-Dimethylformamide (DMF), Piperidine (PIP), trifluoroacetic acid (TFA), dichloromethane (DCM), Acetonitrile (ACN), and dimethyl sulfoxide (DMSO) were purchased from Fisher Scientific International, Inc. Triisopropylsilane (TIPS), thioanizole and N,N-diisopropylethylamine (DIPEA), triphosgene (bis(trichloromethyl) carbonate (BTC), and Triton X-100 were purchased from Sigma-Aldrich. N^α-Fmoc protected amino acids, HBTU ((dimethylamino)-N, N-dimethyl (3H-(1,2,3) triazolo (4,5-b) pyridin-3-yloxy) methaniminium hexafluorophosphate), phenyl silane, tetrakis(triphenylphosphine)palladium(0), and Fmoc-Lys(Boc)-Wang resin (100–200 mesh, 0.32 mmol/g loading capacity) were purchased from Chem-Impex International Inc. Cyanine7.5 NHS ester was purchased from Lumiprobe Corporation. All standard biological reagents were purchased from Sigma-Aldrich and used according to enclosed instructions. Water (18 MΩ) was purified by using a Thermo Scientific Barnstead Smart2Pure Pro water purifying system. Donor horse serum was purchased from Sigma-Aldrich and stored at −20 °C until use in bacterial growth conditions. All necessary precautions as stipulated in the material safety data sheet (MSDS) for each chemical were followed.

Peptide synthesis was carried out on CEM Liberty1 microwave-assisted peptide synthesizer. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Shimadzu system equipped with a CBM-20A communication bus module, two LC-20AT pumps, an SIL-20A autosampler, 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 reflector. Exact mass (EM) data were obtained on an Agilent Technologies 6230 TOF LC/MS spectrometer in positive ion mode and the acceleration voltage on Ion Source I was 19.01 kV. The samples were sprayed with a capillary voltage of 3500 V, and the electrospray ionization (ESI) source parameters were gas temperature of 325 °C at a drying gas flow rate of 3 L/min and a pressure of 25 psi. Beta-galactosidase assays were performed in 96-well plates and measured using a BioTek Synergy H1 multi-mode microplate reader with monochromator-based optics and filter-based optics. All compounds are >95% pure by HPLC analysis.

Peptide synthesis and cyclization:

Peptides were synthesized by solid-phase peptide synthesis using conventional Fmoc chemistry on a pre-loaded Fmoc-L-Lys(Boc)-Wang resin by either a peptide synthesizer or manual synthesis. For the manual synthesis, the resin (0.2 g) was swelled in DCM for 45 min, then peptide synthesis was carried out in DMF. Coupling was conducted using 3 equiv. amino acid compared to the resin loading capacity (0.32–0.4 mmol/g), HBTU as an activating agent, and DIPEA as a base with a 1 h reaction time with shaking (200 rpm). Fmoc deprotection was carried out using 20% PIP in DMF (2×7 min). Following each step, washing with DMF (2×2 min after coupling, 3×2 min after deprotection) was performed. Amino acids bearing alloc-protected amine group on the side chain were added in the selected positions (6 and 10) for future cyclization. For the microwave-assisted synthesis, 0.2 g resin was swelled in DCM, transferred to a reaction vessel, and continued synthesizing the peptide with 0.1 mmol scale synthesis protocol. Initial Fmoc deprotection was carried out with 5 ml of 20% PIP in DMF at 90 °C for 1.5 min at an applied power of 50 W. For each amino acid coupling, 2.5 ml of 0.2 M amino acid stock prepared in DMF, 0.5 ml of 0.5 M HBTU in DMF, and 1 ml of 2 M DIPEA in DMF were mixed and added to the reaction vessel, and the reaction was carried at 75 °C for 5 min at an applied power of 25 W. Amino acids with an alloc-protected amine on the side chain were added manually in the selected positions (6 and 10) for future cyclization using 3 equiv. of amino acid compared to the resin loading capacity. After the last amino acid coupling, the Fmoc group was not removed to keep the N-terminal amino group protected. The successful construction of the linear peptide with alloc-protected amine groups at the 6^th and 10^th positions and an Fmoc-protected N-terminal was confirmed via mass spectrometry prior to cyclization. The alloc-protecting groups were removed using a previously reported protocol.⁴⁷ In brief, the resin was washed three times in DCM for 1 min. At the same time, approximately 5 mL of DCM, dried over 3 Å molecular sieves (dry DCM), was sparged with argon for 2–3 min. Ten equiv. of phenyl silane were then added, and sparging continued for an additional 2–3 min. Then, 0.5 equiv. Tetrakis(triphenylphosphine)palladium(0) was added and sparging continued for additional 5 min. The resulting solution was then added to the resin, and the air in the reaction vessel was displaced with argon before the vessel was sealed with parafilm and then shaken at 200 rpm for 2 h in the dark. The resin was washed with shaking four times with 0.5% sodium diethyldithiocarbamate trihydrate in DMF for 2 min and four times with shaking with DMF for 1 min before proceeding onto the next reaction step. At this point, 50 mg resin was separated for linear peptide preparation, and the remaining 150 mg of resin was used for peptide cyclization (Figure 2D). At first, 0.33 equiv. of BTC dissolved in dry DCM was added to the resin for 30 min at RT with shaking (200 rpm) to form an isocyanate intermediate. Then, 3 equiv. DIPEA were added to complete the cyclization overnight at RT with shaking (200 rpm).⁴⁸ Upon successful cyclization, Fmoc removal was accomplished with the treatment of 5 ml 20% PIP in DMF three times using the microwave-assisted protocol and parameters as follows, 75 °C for 3 min with an applied power of 50 W. The simultaneous side-chain deprotection and cleavage of the peptide from the resin was achieved using 3 ml of a cleavage cocktail containing TFA/TIPS/H₂O at a ratio of 95/2.5/2.5 that was added to the resin for 3 h with shaking. The resin was filtered, and the peptide was precipitated by adding 40 ml cold diethyl ether:hexane (1:1) in a 50 ml centrifuge tube. The precipitated peptide was centrifuged at 4500 rpm (approx. 3840 g) for 5 min. The supernatant was decanted off and the process was repeated to remove the residual cleavage cocktail. The crude peptide pellet was dissolved in ACN:Water (1:1), lyophilized, and purified by RP-HPLC.

Cyanine7.5 labeling:

On resin Cyanine7.5 (Cy7.5) labeling was carried out after the successful synthesis of the desired peptide (CSP1 and CSP1-E1A-cyc(Dab6Dab10)) at the N-terminus. The N-terminal Fmoc protection was removed with 20% PIP and the resulting free amine group was reacted with 0.25 equiv. of amine-reactive derivative of the fluorophore, Cyanine7.5 NHS ester, and 3 equiv. of DIPEA in DMF. The reaction was carried out at RT overnight. Successful labeling was verified by MALDI-TOF MS. The simultaneous side-chain deprotection, cleavage of the peptide from the resin, and further processing was achieved as mentioned above. The labeled and unlabeled peptides were separated and purified by RP-HPLC.

HPLC purification and mass spectrometry analysis:

All the peptides were purified by RP-HPLC using a Phenomenex Kinetex C₁₈ semi-preparative column (5 μm particle size, 10 mm × 250 mm, 110 Å). The linear and cyclized peptides were purified using 18 mΩ H₂O (A)-ACN (B) gradient with 0.1 % TFA. Semi-preparative HPLC methods were used to separate the crude peptide using a linear gradient (first prep 15% B → 55% B over 40 min and second prep 25% B → 35% B over 40 min, flow rate 5 ml/min). In the case of Cy7.5 labeled peptide purification, the linear gradient was as follows: first prep 30% B → 70% B over 55 min and second prep 45% B → 60% B over 40 min, flow rate 5 ml/min. During purification, eluted fractions were verified to contain the desired peptide by MALDI-TOF MS. Then, an analytical HPLC method was used to quantify the purity of the desired product using a linear gradient (5% B → 95% B over 27 min, flow rate 1 ml/min) on a Phenomenex Kinetex analytical C₁₈ column (5 μm particle size, 4.6 mm × 250 mm, 110 Å). The eluted samples containing the desired compound with >95 % purity were pooled, lyophilized, and used for the biological assays. Mass spectrometry analysis was performed on a high-resolution ESI-TOF mass spectrometer to confirm the identity of the peptides (Tables S1–S3).

Bacterial Strain Information:

To assess the ability of the synthesized CSP1-E1A cyclic and linear analogs to modulate the ComD receptors, and thus the QS circuit in S. pneumoniae, β-galactosidase assays were performed using the previously reported D39pcomX∷lacZ (group I) and TIGR4pcomX∷lacZ (group II) reporter strains.³² Phenotypic and in vivo studies were conducting using S. pneumoniae D39 (group 1) and TIGR4 (group 2) strains. For the mixed infection assay, D39-rplL-Erm^R and TIGR4-rplL-Kan^R were constructed and used.

Bacterial Growth Conditions:

Frozen stocks of individual pneumococcal reporter strains were streaked onto a Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) plate containing 5% sterile filtered horse serum and 4 μg/mL concentration of chloramphenicol, and incubated for 8–9 h in a CO₂ incubator (37 °C with 5% CO₂). Freshly grown single colonies were inoculated (single colony for D39pcomX∷lacZ; 2–3 colonies for TIGR4pcomX∷lacZ) into 5 ml of THY broth supplemented with 4 μg/mL final concentration of chloramphenicol and the inoculated culture was incubated in a CO₂ incubator overnight (15 h). The overnight culture was further diluted (1:50 for D39pcomX∷lacZ; 1:10 for TIGR4pcomX∷lacZ) with THY, and the diluted culture was incubated in a CO₂ incubator for 3–4 h. The bacteria were grown until they reached the early exponential stage (0.30–0.35 OD₆₀₀ for D39pcomX∷lacZ; 0.20–0.25 OD₆₀₀ for TIGR4pcomX∷lacZ), as determined by using a plate reader.

Beta-Galactosidase Assays:

The β-galactosidase assays were performed as we previously described.²⁹ See the Supporting Information for full experimental details.

Circular dichroism measurements:

The far-UV circular dichroism (CD) measurements were carried out on an Aviv Biomedical CD spectrophotometer (model 202–01). The peptide secondary structure was evaluated both in an aqueous environment, phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH was adjusted to 7.4), and in membrane mimicking milieu, 20% trifluoroethanol (TFE) in PBS. All the measurements were performed with a peptide concentration of 100 μM or 200 μM, depending on peptide solubility. The far-UV wavelength scans were set between 195 nm – 260 nm and recorded at a speed of 3 nm min⁻¹, with a 20 s data integration time, a data pitch of 1 nm, and using a quartz cuvette of 0.1 cm path length. In all experiments, the observed high-tension value was ~650 V at the shortest wavelength of 195 nm at 25 °C. The single scan was acquired, corrected for the respective solvent contributions, and converted to mean residue ellipticity (MRE) values using the following equation:

$$MRE=\left(\frac{\theta }{10\times c\times l}\right)/n$$

Where, θ is the observed ellipticity in millidegrees, c is the peptide concentration in molarity, l is the path length in centimeters, and n is the length of the peptide sequence.

Percent helicity (f_H) was calculated for peptides that exhibited a significant helical pattern using the following equation:

$$fH=\frac{{[\theta ]}_{222}}{{[\theta \infty ]}_{222}\left(1-\frac{x}{n}\right)}$$

Where, [θ]₂₂₂ is the mean residue ellipticity of the sample peptide at 222 nm, [θ_∞]222 is the mean residue ellipticity of an ideal peptide with 100% helicity (−44 000 deg cm² dmol⁻¹), n is the number of residues in the potential helical region, and x is an empirical correction for end effects (2.5).

Western blot detection of released PLY in culture supernatant:

Pneumococcal strains were streaked on THY agar to isolate a single colony, which was then inoculated into THY liquid medium and cultured at 37 °C with 5% CO₂ for monoclonal expansion. At OD_600nm=0.2, varying concentrations of CSP1-E1A-cyc(Dab6Dab10) were added to 25 mL cultures of D39 and TIGR4 together with CSP1 or CSP2 (45 or 46 nM), respectively. Sterile PBS of equal volume was used as a control. After 30 min of incubation, broth cultures were centrifuged at 3000 × g for 15 min at 4 °C to collect the supernatants. Collected supernatants were filtered through 0.22 μm syringe filters (Millipore). Cell-free supernatants containing PLY (53 KDa) were concentrated 50x by using the 30 KDa filter unit (AMICON), by following manufacturer’s instructions. The concentrated supernatants were serially diluted with fresh THB for western-blot detection by using a primary antibody specific for PLY (Santa Cruz Biotechnology sc-80500, 1:1000 dilution). Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2005, 1:10,000 dilution) was used as a secondary antibody, and HRP label was detected by an ECL substrate (Bio-Rad, 170–5060).

Hemolysis assay:

Hemolytic assays were performed as we have previously published.^41–42 Serial two-fold dilutions of PLY-containing supernatants from the aforementioned western-blot analysis were incubated with 10 mM dithiothreitol (DTT) at room temperature for 15 min before performing the hemolysis assay. An aliquot of each sample (in 500 μl) was mixed with 200 μl of 2% sheep red blood cells and incubated for 30 min at 37 °C. The mixture was centrifuged at 3,000 × g for 5 min. The absorbance of the cell-free supernatant was measured at OD 541_nm. The hemolytic units (HU) were reported as the highest dilution that could lyse 50% of the red blood cells (Figure S13).

Mouse mortality assay

Seven-week old CD-1 mice (cohorts of 15, males and females) were anesthetized with isoflurane and intranasally administered with either D39 (4.5 × 10⁶ CFU) or TIGR4 (1.07 × 10⁶ CFU). Infected mice were intravenously (retro-orbital) treated with 4 mg/kg of CSP1-E1A-cyc(Dab6Dab10) at 2 hpi and 24 hpi. Mouse mortality was monitored for 120 hpi. Moribund animals that displayed the following symptoms, including severe lethargy, roughened fur, hunch posture, distended abdomen, inflamed and closed eyes and inability to feed were euthanized. Times of death or euthanasia were recorded accordingly.

Murine model of acute pneumonia mixed infection and subsequent invasive spread

Seven-week-old CD-1 mice (cohorts of 10, males and females) were intranasally inoculated with a 1:1 mixture (2.5 × 10⁶ CFU/strain) of D39-rplL-Erm^R and TIGR4-rplL-Kan^R (total dose of 5 × 10⁶ CFU). Infected mice were intravenously (retro-orbital) treated with 4 mg/kg of CSP1-E1A-cyc(Dab6Dab10) or PBS at 2 hpi and 24hpi. Animals were euthanized at 48 hpi, and lungs and spleens were homogenized and serially diluted in sterile saline, and plated in a non-selective THY agar, THY agar supplemented with erythromycin (250 ng/mL) or Kanamycin (200 ng/mL) to assess the burden of each bacterial strain in each organ.

In vivo imaging

Seven-week-old CD-1 mice (cohorts of 4–5) were intravenously (retro-orbital) administered with CSP1-Cy7.5 or CSP1-E1A-cyc(Dab6Dab10)-Cy7.5 (50 μg/mouse) and subjected to anesthesia by 3% isoflurane within an induction chamber. Mice were imaged with the IVIS SpectrumCT imaging system (Perkin-Elmer, Waltham, MA). Fluorescence signals were captured using the ex/em value of 745/800 and images were acquired in the settings of binning factor=4, f number of 1, field view at 25.4 cm. The acquired images were analyzed by the Living Image software (Perkin-Elmer).

Ethics statement

Mouse studies conducted in this study strictly followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign (protocol 21086).

ABBREVIATIONS:

ACN

acetonitrile

AA

amino acid

BTC

(bis(trichloromethyl) carbonate

CD

Circular Dichroism

CFU

colony forming unit

CSP

competence stimulating peptide

Cy7.5

Cyanine7.5

Dab

2,4-diaminobutyric acid

Dap

2,3-diaminopropionic acid

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

dnCSP

Dominant Negative CSP

DMF

N,N-Dimethylformamide

DTT

dithiothreitol

ESI

electrospray ionization

HBTU

(dimethylamino)-N, N-dimethyl (3H-(1,2,3) triazolo (4,5-b) pyridin-3-yloxy) methaniminium hexafluorophosphate

hpi

hours post infection

HU

hemolytic units

IVIS

in vivo imaging system

MALDI-TOF MS

matrix-assisted laser desorption ionization time-of-flight mass spectrometry

MRE

mean residue ellipticity

OD

optical density

Orn

ornithine

PBS

phosphate-buffered saline

PIP

piperidine

PLY

pneumolysin

PMF

proton motive force

QS

quorum sensing

RP-HPLC

reversed-phase high-performance liquid chromatography

RT

room temperature

SAR

structure-activity relationship

SPPS

solid-phase peptide synthesis

TCSTS

two-component signal transduction system

TFA

trifluoroacetic acid

TFE

trifluoroethanol

THY

Todd–Hewitt broth with yeast extract

TIPS

triisopropylsilane

UV

ultraviolet