Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

Abstract

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.

Figure 1. Amino acid sequences of CSP1 and CSP2, and their analogues.

Modified CSPs were synthesized based on the sequence of CSP1 or CSP2 by amino acid substitutions or by deletion. The ability of each CSP analogue to induce competence and to competitively inhibit CSP1 or CSP2-mediated competence is presented. *No represents < 1% of CSP1 or CSP2 activity during the induction of competence.

Figure 2. The first and third amino acid residues of CSP1 are essential for induction of competence.

(A) D39pcomX::lacZ cells were incubated with CSP1 or with individual modified peptides at a final concentration of 100 ng/ml for 30 min. The ability of each peptide to induce competence regulon was measured by induction of the comX promoter in D39pcomX::lacZ. The amino acid substitution of 1^st amino acid (CSP1-E1A) and 3^rd amino acid (CSP1-R3A) in the N-terminus abolished the peptide's ability to induce the expression of comX. (B) D39 cells were exposed to 100 ng/ml of CSP1 or individual modified peptides. DNA transformation was performed using the rpsL gene. Transformants were selected on THB agar containing 100 µg/ml streptomycin. Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. *p<0.01 when compared against CSP1-treated cells.

Figure 3. Dose dependent inhibition of comX expression and DNA transformation by CSP-E1A.

(A–B) CSP-E1A but not CSP1-R3A inhibits CSP1-mediated induction of comX in a dose dependent manner. D39pcomX::lacZ cells were exposed to 100 ng/ml of CSP1 alone or simultaneously with increasing concentrations of CSP-E1A or CSP-R3A. The activity of comX gene promoter was measured by b-galactosidase activity. (A) CSP-E1A competitively inhibits the ability of CSP1 to induce comX. (B) CSP-R3A is unable to induce comX, and does not competitively inhibit CSP1. (C–D) CSP-E1A but not CSP1-R3A outcompetes and inhibits CSP1-mediated genetic transformation in a dose dependent manner. In vitro transformation was performed by treating D39 with 100 ng/ml CSP1 alone or simultaneously with increasing concentrations of CSP1-E1A or CSP1-R3A. Purified PCR product harboring a mutated rpsL gene was used for genetic transformation. Transformants were selected on THB agar plates supplemented with 100 µg/ml streptomycin. CSP1-E1A competitively inhibited the ability of CSP1 to induce DNA transformation (C). CSP1-R3A was unable to competitively inhibit DNA transformation (D). Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. *p<0.01 when compared against cells treated with CSP1 only.

Figure 4. Time-dependent inhibition of CSP1 by CSP1-E1A.

CSP1-E1A competitively inhibits CSP1 in a time-dependent manner. D39pcomX::lacZ cells (OD 600_nm 0.1) were pre-exposed to 100 ng/ml CSP1. At Time 0, 5, 10, 15, 20, 25 and 30 min, CSP-E1A (100 ng or 800 ng) was added to chase CSP1. Cells were collected 30 min after each addition of CSP1-E1A for b-galactosidase assays. Activation of comX promoter was calculated against D39pcomX::lacZ cells exposed to CSP1 alone (100%). Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown.

Figure 5. CSP1-E1A delays and inhibits the spontaneous development of competence in S. pneumoniae in a concentration-dependent manner.

(A–B) D39pcomX::lacZ and D39pcbpD::lacZ cells grown in THB (pH 8.3) were incubated with 100 ng/ml, 1 µg/ml or 10 µg/ml of CSP1-E1A or with same volume of sterile water, and monitored for comX promoter activity (A) or for the cbpD promoter activity (B) by b-galactosidase assays at indicated time intervals. (C) CSP1-E1A does not inhibit the growth of S. pneumoniae. Similar results were obtained from three independent experiments. The data from one typical experiment are shown.

Figure 6. CSP1-E1A inhibits the expression of competence regulated virulence factors LytA and CbpD in vitro.

(A) CSP1-E1A attenuates the expression of LytA and CbpD. D39 cells (OD 600_nm 0.1) were exposed to CSP1 (100 ng/ml) alone or simultaneously to increasing amounts of CSP1-E1A (0, 100, 200, 400 or 800 ng/ml). Cells were incubated for 30 minutes, lysed and subjected to zymogram analysis. Lytic activity was observed as bands of clear zones in the turbid gel. Experiments were repeated three times. The gel from one typical experiment is shown. (B–C) Densitometry analysis of LytA (B) and CbpD (C) from the zymogram in A. (D) CSP1-E1A inhibits the promoter activity of cbpD in a concentration dependent manner. D39pcbpD::lacZ cells were exposed to CSP1 alone, pure water alone or to a mixture of 100 ng/ml CSP1 and CSP-E1A.Cells were collected for b-galacosidase assays 30 min after exposure.

Figure 7. CSP1-E1A attenuates the mortality of mice infected with S. pneumoniae.

(A) Six-week old CD-1 mice (n = 10) were intranasally infected with S. pneumoniae D39 (3×10⁶, 50 µl) alone, or with a mixture of D39-CSP-E1A (20 µg/mouse). Infected mice were monitored every 6 hr. Moribund mice were euthanized and considered dead. The difference in kinetics of mouse survival between the two groups was not statistically significant (p = 0.21) as analyzed by the log rank (Mantel-Cox) survival analysis. (B) Infection assay was repeated with CD-1 mice (n = 20) using D39 (2×10⁶, 50 µl) alone or with a mixture of D39-CSP-E1A (100 µg/mouse). Infected mice were monitored as described in (A). The kinetics of death was significantly delayed (p = 0.01) as determined by the log rank (Mantel-Cox) survival analysis. (C) The survival kinetics was indistinguishable between mice infected with D39 alone or in mice infected with a mixture of D39 and 100 µg CSP1-R3A (p = 0.96).

Figure 8. CSP2-E1A inhibits competence development and transformation in ComD2 strain TIGR4.

(A–B) CSP2-E1A was drastically reduced in its ability to induce the promoter activity of comX (A) and DNA transformation in TIGR4 (B). Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. *p<0.01 when compared against TIGR4pcomX::lacZ cells (A) or TIGR4 cells (B) treated with CSP2 only. (C) CSP-E2A out-competes CSP2 and inhibits activation of the comX promoter activity. TIGR4pcomX::lacZ cells (OD 600_nm 0.1) were incubated with 100 ng/ml CSP2 alone or simultaneously with increasing amounts of CSP2-E1A. The activity of comX promoter was measured by b-galactosidase assays. (D) CSP-E2A inhibits CSP2-mediated DNA transformation of TIGR4. In vitro transformation was performed by treating TIGR4 cells with 100 ng/ml CSP2 alone or simultaneously with increasing amount of CSP2-E1A. Purified PCR product of a mutated rpsL gene was used for genetic transformation. Transformants were selected on THB agar supplemented with 100 µg/ml streptomycin. Experiments in were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. *p<0.01 when compared against TIGR4pcomX::lacZ cells (A) or TIGR4 cells (B) treated with CSP2 only.

Figure 9. Cross inhibition of competence by CSP1-E1A and CSP2-E1A in S. pneumoniae strains with incompatible ComD subtypes.

(A-B) TIGR4 (ComD2 subtype) or TIGR4pcomX-lacZ cells (OD 600_nm 0.1) were incubated with CSP2 alone or simultaneously with increasing amounts of CSP1-E1A (100–800 ng/ml). (C-D) D39 (ComD1 subtype) or D39pcomX-lacZ cells were incubated with the mixture of 100 ng/ml CSP1 alone or simultaneously with increasing amounts of CSP2-E1A (100–800 ng/ml). Pneumococcal cells were assayed for ß-galactosidase activity and transformation frequency 60 minutes after exposure. (A–B) Cross inhibition of competence in ComD2 strain TIGR4 by CSP1-E1A as measured by the induction of comX promoter activity (A) and transformation frequency (B). (C–D) Cross inhibition of competence in ComD1 strain D39 by CSP2-E1A as measured by the induction of comX promoter activity (C) and transformation frequency (D). Experiments were performed in triplicates and repeated three times. The means ± SD of one typical experiment are shown. *p<0.05 when compared against TIGR4 cells or TIGR4pcomX-lacZ cells treated with CSP2 alone, or D39 cells or D39pcomX-lacZ cells treated with CSP1 alone.