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. 2022 Apr 20;5(5):299–305. doi: 10.1021/acsptsci.2c00037

Pharmacological Evaluation of Synthetic Dominant-Negative Peptides Derived from the Competence-Stimulating Peptide of Streptococcus pneumoniae

Myung Whan Oh §, Muralikrishna Lella ‡, Shanny Hsuan Kuo §, Yftah Tal-Gan ‡,*, Gee W Lau §,*
PMCID: PMC9112410  PMID: 35592433

Abstract

graphic file with name pt2c00037_0005.jpg

The competence regulon of Streptococcus pneumoniae (pneumococcus) is a quorum-sensing circuitry that regulates the ability of this pathogen to acquire antibiotic resistance or perform serotype switching, leading to vaccine-escape serotypes, via horizontal gene transfer, as well as initiate virulence. Induction of the competence regulon is centered on binding of the competence-stimulating peptide (CSP) to its cognate receptor, ComD. We have recently synthesized multiple dominant-negative peptide analogs capable of inhibiting competence induction and virulence in S. pneumoniae. However, the pharmacodynamics and safety profiles of these peptide drug leads have not been characterized. Therefore, in this study, we compared the biostability of cyanine-7.5-labeled wild-type CSPs versus dominant-negative peptide analogs (dnCSPs) spatiotemporally by using an IVIS Spectrum in vivo imaging system. Moreover, in vitro cytotoxicity and in vivo toxicity were evaluated. We conclude that our best peptide analog, CSP1-E1A-cyc(Dap6E10), is an attractive therapeutic agent against pneumococcal infection with superior safety and pharmacokinetics profiles.

Keywords: Streptococcus pneumoniae, competence regulon, genetic transformation, virulence, competence stimulating peptide, peptide therapeutics

Streptococcus pneumoniae (pneumococcus) commonly inhabits the human nasopharyngeal tract as a commensal, but it can become a significant opportunistic pathogen in children, the elderly, and immunocompromised individuals, resulting in a variety of diseases ranging from mild respiratory infection to acute otitis media, community-acquired pneumonia, invasive bacteremia, pneumonia-derived sepsis, and meningitis.1,2 Pneumococcus is a model organism used to unravel various molecular and cellular mechanisms underlying DNA uptake and genetic transformation. Through competence induction, a unique physiological state, pneumococci actively scavenge for extracellular DNA from non-competent sister cells and exchange DNA with closely related species occupying the same host niche.3,4 Pneumococcal competence is centered on a 17-amino acid quorum-sensing (QS) peptide called the competence-stimulating peptide (CSP), with two major variants that cover the majority of pneumococcal serotypes: CSP1 and CSP2.5 During growth, pneumococcal cells continuously secrete CSP to the extracellular environment. When the accumulated CSP reaches threshold levels, the diffusive pheromone peptide binds to and activates the ComDE two-component regulatory system, resulting in upregulation of genes involved in DNA uptake and transformation.6 An alternative model of competence activation posits that competent pneumococcal cells spread QS signal via cell-to-cell contact whereby CSP peptides bound to its cell surface ComD receptor are transmitted to other cells.7,8 Regardless of the model of activation, subsequently, the alternative sigma factor, ComX, is upregulated. ComX then binds to “combox”-containing promoters and initiates the transcription of approximately 90 “late” genes that are important for genetic transformation9,10 and virulence.11

Among the “late” competence genes are those encoding the allolytic factors, CbpD, LytA, and CibAB,3,10,11 which attack the non-competent neighboring pneumococcal cells and/or closely related streptococcal species and promote the release of DNA, lipoteichoic acid (LTA), and pneumolysin (PLY), a cholesterol-dependent cytolysin toxin.12,13 It is likely that PLY released by the action of CbpD, LytA, and CibAB damages the alveolar–capillary barrier,3,11 allowing pneumococcus to invade the bloodstream to cause sepsis.8

Antibiotic resistance is a major problem in modern human medicine. Multi-drug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) pneumococcal strains are emerging rapidly, dwindling our choices of available drug options against this pathogen.14−16 These resistant traits are driven primarily by competence regulon-mediated horizontal gene transfer and integrative and conjugative elements localized to genomic islands.17,18 While immunization with conjugated vaccines reduces the carriage of antibiotic-resistant pneumococcal serotypes, they impose selection pressure for serotype switching, resulting in vaccine-evading serotype MDR strains with elevated resistance to commonly prescribed antimicrobials, including β-lactams and macrolides.19,20 Moreover, fluoroquinolone antibiotics have been reported to activate pneumococcal competence, which may inadvertently exacerbate the expression of allolytic factors that enhance PLY release.21 Thus, alternative therapeutic strategies that do not impose selection pressure on the emergence of antibiotic resistance and serotype switching are urgently needed.

Because the competence regulon is important for horizontal gene transfer and regulates pneumococcal virulence,3,8,11,22−24 we have developed dominant-negative competence-stimulating peptides (dnCSPs) and successfully demonstrated that these peptide analogs competitively interfere with CSP-ComD interactions, inhibit competence induction, reduce the acquisition of antibiotic resistance and capsule biosynthesis genes, decrease the expression of the allolytic factors LytA and CbpD and the resulting release of PLY, and attenuate mouse mortality during acute pneumonia infections.25−27 However, to date, the pharmacological properties and safety profiles of these dnCSPs have not been examined. Therefore, herein we investigated the biostability of CSPs and dnCSPs in vivo spatiotemporally by live imaging. Furthermore, we compared the in vitro cytotoxicity toward lung epithelial cells and in vivo toxicity of these peptides by analyzing serum chemistry, hematology, and histopathology in mice. Our results indicate that our lead dnCSPs are promising drug candidates against pneumococcal infections with strong pharmacological profiles.

Results and Discussion

In this work we evaluated how peptide cyclization affects key pharmacological properties of lead dnCSPs. To this end, we performed in-depth analysis of metabolic stability and in vivo localization of lead CSPs: the two natural CSPs (CSP1 and CSP2), lead linear dnCSPs (CSP1-E1A, CSP2-E1Ad10),25,27,28 and a lead cyclic dnCSP (CSP1-E1A-cyc(Dap6E10)).26 To achieve that, we designed and constructed a set of Cyanine7.5 (Cy7.5)-labeled CSP analogs where activated Cy7.5 fluorophore was attached to the N-terminus of the peptides (Figure 1A,B). Detailed descriptions of dnCSP synthesis and fluorescence labeling can be found in the Supporting Information.

Figure 1.

Figure 1

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CSP analogs synthesis and Cy7.5 labeling methodology. (A) Linear or cyclic peptides (see Table S1) were successfully synthesized using solid-phase peptide synthesis and confirmed by MALDI-TOF-MS. N-terminal Fmoc was removed, and Cy7.5 labeling was performed using activated cyanine NHS ester (0.25 equiv for linear peptides and 0.5 equiv for cyclic peptides) in DMF with 3 equiv of DIPEA. After successful labeling, the peptide was cleaved from the resin, and the labeled and unlabeled peptides were separated and purified by RP-HPLC. (B) Detailed chemical structures of Cy7.5-labeled CSPs. Here, E1A represents that Glu at the first position is replaced by Ala, Dap represents 2,3-diaminopropionic acid, and d represents d-aspartic acid (d-Asp).

Cy7.5-CSP1, Cy7.5-CSP1-E1A, Cy7.5-CSP1-E1A-cyc(Dap6E10), Cy7.5-CSP2, and Cy7.5-CSP2-E1Ad10 were intravenously (i.v.) administered at 50 μg concentration to CD-1 mice, and the fluorescence outputs were monitored spatiotemporally by using an IVIS SpectrumCT in vivo imaging system (PerkinElmer), as we have previously described,8,29 with excitation/emission values set at the 745/800 nm. Note that although we were successful in labeling CSP2, we encountered issues in solubilizing the Cy7.5-CSP2 analog in the aqueous conditions required for the IVIS studies. Compared to CSP1, CSP2 is significantly more hydrophobic and tends to adopt a β-sheet conformation, which is susceptible to aggregation. When coupled with the hydrophobic Cy7.5 label, the Cy7.5-CSP2 analog was too hydrophobic to effectively dissolve in the aqueous buffer, resulting in this analog precipitating out of solution, and no fluorescence signal was detected in mice (data not shown). As we have previously shown, a single d-amino acid substitution in the 10th position (d10) of CSP2 results in a significant conformational change, from a β-sheet to an α-helix conformation, increasing dramatically the solubility of the peptide analog (CSP2-d10) and any other peptide variants bearing this modification.30 For the remainder dnCSPs, injected Cy7.5-labeled peptides immediately distributed throughout the entire mouse (Figure 2). Cy7.5-CSP1 inoculated mice exhibited systemic strong signal outputs until 6 hours post inoculation (hpi), fading greatly by 12 hpi and with only minute residual amounts visible by 24-hpi (Figure 2). Mice injected with the two labeled linear dnCSPs, Cy7.5-CSP1-E1A and Cy7.5-CSP2-E1Ad10, displayed longer systemic fluorescence signal outputs until 12 hpi, but began to fade by 24 hpi, and only a residual amount of signal was detected at 36 hpi (Figure 2). These observations suggest that biodegradation of native CSP1 occurred at a significant amount between 6 and 12 hpi, whereas the biodegradation of CSP1-E1A and CSP2-E1Ad10 took place later, between 12 and 24 hpi, indicating that these linear dnCSPs have longer half-lives than the native CSP. Also, the intensities of CSP2-E1Ad10 signals were generally weaker than Cy7.5-CSP1-E1A. Importantly, Cy7.5-CSP1-E1A-cyc(Dap6E10) demonstrated strikingly enhanced stability compared to the Cy7.5-CSP1 as well as both linear dnCSPs, with strong fluorescence signals that lasted over 175 hpi in all mice, with mice D2, D3, and D4 retaining systemic signal output until 96 hpi, while mice D1 and D5 retained systemic signal output until 60 hpi (Figure 2). Overall, our results indicate a far superior biostability of CSP1-E1A-cyc(Dap6E10) as a result of macrocyclization of the peptide compared to linear native CSP and dnCSPs, providing higher therapeutic potential against pneumococcal infections as we have demonstrated recently.26

Figure 2.

Figure 2

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CSP1-E1A-cyc(Dap6E10) exhibits superior biostability compared to native CSP1 and linear analogs. Cyanine7.5 (Cy7.5)-labeled CSPs or dnCSPs were intravenously administered into CD-1 mice (n = 4–5, 50 μg) via a retro-orbital route. The biostability of native Cy7.5-CSP1 and dnCSPs (Cy7.5-CSP1-E1A, Cy7.5-CSP2-E1Ad10, and Cy7.5-CSP1-E1A-cyc(Dap6E10)) was imaged at the indicated time points by using an IVIS SpectrumCT imaging system. The excitation/emission parameters of IVIS were set to 745 nm/800 nm.

To be therapeutically viable, various dnCSPs have to be non-cytotoxic. Thus, we examined the cytotoxicity of both native and dnCSPs. The WST-1 cell proliferation and viability assay (Roche Molecular Systems, Indianapolis, IN, USA) analyzes the number of viable cells by the cleavage of tetrazolium salts, and expansion of viable cells increases the overall activity of mitochondrial dehydrogenases in the cell culture, which, in turn, increases the amount of formazan dye measurable by absorbance using a spectrophotometer at OD480 nm. The cytotoxicity of each dnCSP along with both native CSP1 and CSP2 was examined in the airway bronchial epithelial 16HBE cells and the lung carcinoma alveolar type 2-like A549 cells. Both lung epithelial cells were challenged with two different doses (50 or 100 μg mL–1) of CSP1, CSP2, or individual dnCSPs for 24 h. Importantly, both native CSP1 and CSP2 as well as their derivatives did not significantly alter the viability of 16HBE cells compared to the PBS control except for a slight decrease in viability after exposure to 100 μg mL–1 of CSP1-E1A and CSP2-E1Ad10 (Figure 3). Similarly, cell viability of A549 cells was unaffected by exposure to these peptides (Figure 3).

Figure 3.

Figure 3

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In vitro cytotoxicity of dnCSPs. Bronchial epithelial 16HBE cells and alveolar type 2-like lung carcinoma A549 cells seeded in a 96-well cell culture plate were exposed to 50 or 100 μg mL–1 of native CSP1 or CSP2, or each dnCSP for 24 h. Cytotoxicity was determined by using the WST-1 cell proliferation and viability assays, and absorbance was measured at OD480 nm. Error bars represent SEM for n = 8–16. Significance was determined with one-way ANOVA with Bonferroni’s correction against the PBS control. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control; ns, not significant.

We have shown that the dnCSP CSP1-E1A-cyc(Dap6E10) has high efficacy in protecting against acute pneumonia,26 has superior biostability against proteolytic degradation in mice (Figure 2), and is non-cytotoxic toward lung epithelial cells. Additionally, intranasally administered CSP1-E1A-cyc(Dap6E10) did not show any overt toxicity in mouse lungs.26 To determine potential systemic toxicity of CSP1-E1A-cyc(Dap6E10), we focused our safety analysis on the cyclic dnCSP in vivo by analyzing the mouse weight (Figure 4A), histopathology of vital organs (Figure 4B–F), blood serum chemistry (Table 1), and hematology (Table 2) after intraperitoneal administration of CSP1-E1A-cyc(Dap6E10) at mouse therapeutic dose (4 mg kg–1) daily over 6 days. The weight of mice treated with CSP1-E1A-cyc(Dap6E10) was indistinguishable to the PBS control cohort (Figure 4A). Histopathological analysis of hematoxylin and eosin stained tissue sections from lungs, spleens, hearts, kidneys, and livers revealed that exposure to CSP1-E1A-cyc(Dap6E10) did not elicit significant changes in these vital organs compared to the PBS treatment (Figure 4B–F). Mild hepatocellular cytoplasmic vacuolations were observed in the livers of both CSP1-E1A-cyc(Dap6E10) and PBS control cohorts, indicating no treatment-specific changes.

Figure 4.

Figure 4

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CSP1-E1A-cyc(Dap6E10) is not toxic in vivo. Six-week-old CD-1 mice (n = 5 per cohort) were exposed to CSP1-E1A-cyc(Dap6E10) (4 mg kg–1) or sterile PBS once daily for 6 days. (A) Mouse weight over the course of exposure. (B–F) Histopathology of major organs after the dnCSP or PBS challenge. Organ sections were stained with hematoxylin and eosin.

Table 1. Blood Serum Chemistry of Mice after Exposure to CSP1-E1A-cyc(Dap6E10).

analyte saline CSP1-E1A-cyc(Dap6E10) reference range
creatinine 0.20 ± 0.10 0.16 ± 0.05 0.1–0.4
BUN (urea) 24 ± 2.65 21.00 ± 2.00 16–29
total protein 5.03 ± 0.35 4.80 ± 0.11 5.0–6.3
albumin 2.57 ± 0.12 2.54 ± 0.08 3.0–4.1
globulin 2.53 ± 0.32 2.26 ± 0.08 1.8–2.3
albumin/globulin ratio 1.13 ± 0.21 1.12 ± 0.07  
calcium 10.27 ± 0.21 10.22 ± 0.23 9.8–10.8
phosphorus 7.77 ± 1.80 8.68 ± 1.37 6.1–13.1
sodium 152.00 ± 3.46 157.00 ± 1.67 153–159
potassium 6.30 ± 2.88 4.12 ± 0.39 8.3–12.7
sodium/potassium ratio 27.67 ± 12.01 38.40 ± 4.63  
chloride 112.00 ± 2.65 110.40 ± 0.80 106.1–113.9
glucose 145.33 ± 27.43 116.00 ± 6.04 169–298
alkaline phos total 94.00 ± 67.20 155.00 ± 40.58 34–106
ALT (SGPT) 50.67 ± 11.59 58.00 ± 18.28 25–76
GGT 0.00 ± 0.00 0.00 ± 0.00 36–89
total bilirubin 0.37 ± 0.12 0.26 ± 0.05 0.16–0.31
cholesterol total 159.67 ± 15.53 152.20 ± 14.77 111–196
triglycerides 156.33 ± 38.07 116.80 ± 42.01 51–161
bicarbonate (TCO2) 7.67 ± 1.53 8.60 ± 1.50 N/Aa
anion gap 39.00 ± 3.00 42.00 ± 3.03 N/A
lipemic indicator 0.00 ± 0.00 0.00 ± 0.00 N/A
icteric indicator 0.00 ± 0.00 0.00 ± 0.00 N/A
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a

N/A: not available.

Table 2. Complete Blood Count with Differential of CD-1 Mice after Exposure to CSP1-E1A-cyc(Dap6E10).

analyte saline CSP1-E1A-cyc(Dap6E10) reference range
red blood cells (×106/mL) 8.38 ± 0.64 7.85 ± 0.32 7.31–12.27
hemoglobin (g/dL) 13.67 ± 0.77 13.06 ± 0.69 11.9–18.4
hematocrit (%) 40.25 ± 2.35 39.68 ± 2.02 39.7–74.7
mean cell volume (fL) 48.13 ± 1.36 50.56 ± 1.28 46.5–69.0
MCH (pg) 16.33 ± 0.30 16.66 ± 0.42 13.1–18.0
MCHC (g/dL) 33.93 ± 0.81 32.90 ± 0.23 21.3–33.9
platelet estimate (×103/mL) 166.20 ± 93.62a 560.00 ± 251.32a 736–2374
WBC count (×103/mL) 6.50 ± 1.67 4.28 ± 0.94a 4.44–14.01
Neu (%) 17.10 ± 12.00 14.34 ± 8.89 8.74–55.68
lymph (%) 76.37 ± 16.82 79.98 ± 12.15 37.50–85.01
mono (%) 5.02 ± 5.27 4.18 ± 2.95 2.84–13.09
Eos (%) 1.48 ± 0.50 0.62 ± 0.57 0.30–5.20
Baso (%) 0.03 ± 0.05 0.08 ± 0.18 0.01–1.70
A Neu (×103/mL) 1.26 ± 1.08 0.62 ± 0.48 0.53–5.17
A Lymph (×103/mL) 4.76 ± 0.26 3.41 ± 0.91 2.06–10.01
A Mono (×103/mL) 0.38 ± 0.46 0.18 ± 0.17 0.18–1.32
A Eos (×103/mL) 0.11 ± 0.06 0.02 ± 0.02 0.01–0.83
A Baso (×103/mL) 0.00 ± 0.01 0.00 ± 0.00 0.00–0.17
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a

Below the reference range.

There were no significant differences between mouse cohorts treated with CSP1-E1A-cyc(Dap6E10) versus the PBS in profiles of both blood chemistry (Table 1) as well as hematological analysis by complete blood count with differential (Table 2). Blood serum biochemistry revealed a mild degree of hypoalbuminemia and hypoglycemia in both control and CSP1-E1A-cyc(Dap6E10) cohorts. Also, there was slightly elevated total bilirubin in the control group, which may indicate a mild portal triaditis (Table 1). In the hematological analysis, low platelet count was observed in both control and CSP1-E1A-cyc(Dap6E10) cohorts, most likely caused by common clotting events during blood collection process (Table 2). In the hematological analysis, the total white blood cell count in the CSP1-E1A-cyc(Dap6E10) mice was minimally below the normal reference range. However, the mean cell volume, MCHC, and eosinophil count resulting from the CSP1-E1A-cyc(Dap6E10) treatment were within the normal reference ranges. Similarly, the measurements of WBC subsets such as lymphocyte, neutrophil, monocyte, eosinophil, and basophil were within the normal reference range. These hematological analyses indicate that CSP1-E1A-cyc(Dap6E10) is non-pathological. Because these mild outliers are also found in the control cohort, we concluded that systemic exposure to CSP1-E1A-cyc(Dap6E10) within the duration of treatment window during lung infection by pneumococcus does not cause major abnormalities.

In summary, the cyclic dnCSP CSP1-E1A-cyc(Dap6E10) shows promising pharmacological properties. The dnCSP demonstrates prolonged biostability against proteolytic degradation in vivo. It is also non-cytotoxic toward two lung epithelial cell lines in vitro. Histopathology of various vital organs, blood chemistry, and hematological analyses revealed no abnormality, further bolstering the safety and utility of CSP1-E1A-cyc(Dap6E10) as a new therapeutic that does not place a selective pressure on pneumococcus for resistance development.

Acknowledgments

We thank D. C. Gruenert (University of California, San Francisco, CA, USA) for the gift of 16HBE cells. This work was supported by the NIH (HL142626) to Y.T. and G.W.L.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00037.

  • Experimental methods, HPLC traces for CSP analogs, and Table S1, listing MS and HPLC data for CSP analogs (PDF)

Author Contributions

† M.W.O. and M.L. contributed equally to this work.

The authors are solely responsible for experimental designs and data analysis. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.

Supplementary Material

pt2c00037_si_001.pdf (434.2KB, pdf)

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