Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide

Abstract

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S. mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S. mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S. mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S. mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S. mitis CSP signal can modulate QS response in S. pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S. mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S. mitis as it inhabits its natural niche.

Keywords: Streptococcus mitis; Streptococcus pneumoniae; Structure-activity relationships; competence stimulating peptide (CSP); quorum sensing.

Figure 1

General streptococcal CSP-mediated QS pathway. ComC is processed and exported to the extracellular environment as the mature CSP signal by ComAB. At high concentration, the CSP binds to its cognate histidine-kinase receptor ComD. Activation of ComD leads to phosphorylation of ComE, a response regulator, resulting in further activation of the comABCDE operon and expression of comX, the master regulator of group-behavior genes. The sequences of the Streptococcus pneumoniae CSP1 and CSP2 as well as Streptococcus mitis-CSP-2 are presented at the top. CSP, competence-stimulating peptide; QS, quorum sensing.

Figure 2

Predicted S. mitis-CSP-2 sequence based on sequencing results.A, ClustalW alignment of the comC gene product of several mitis group streptococci. ComC is typically cleaved at the double glycine site to afford a mature CSP signal with a negatively charged N-terminal residue. B, primers used for comC amplification. C, identification of Streptococcus mitis-CSP-2 following comC sequencing. CSP, competence-stimulating peptide.

Figure 3

Isolation and detection of the Streptococcus mitis-CSP-2 from cell-free supernatants. The RP-HPLC chromatogram of total proteins isolated from the supernatant sample and high-resolution ESI-TOF MS of the fraction collected from 28 to 30 min (red). See the Supporting information for full experimental details. CSP, competence-stimulating peptide.

Figure 4

Comparison of purified synthetic and isolated Streptococcus mitis-CSP-2.A, proposed structure of the 16-amino acid S. mitis-CSP-2. B, comparison of observed masses of isolated and synthetic peptides by ESI-TOF MS. C, comparison of analytical RP-HPLC chromatograms of the purified isolated, synthetic, and isolated and synthetic S. mitis-CSP-2. CSP, competence-stimulating peptide.

Figure 5

Structure of Streptococcus mitis-CSP-2 highlighting the SAR trends observed throughTable 1, Table 2, Table 3. CSP, competence-stimulating peptide; SAR, structure-activity relationship.

Figure 6

Structures of Streptococcus mitis-CSP-2 and S. mitis-CSP-2-N7II8F with the modified residues highlighted. CSP, competence-stimulating peptide.

Figure 7

Transformation assay of Streptococcus mitis ATCC 49456 in the presence of S. mitis-CSP-2, S. mitis-CSP-2-n11, and S. mitis-CSP-2 together with S. mitis-CSP-2-E1Af10r16. The ability of ATCC 49456 to internalize a spectinomycin-resistance plasmid (pFW5-luc) was evaluated following treatment with S. mitis-CSP-2, S. mitis-CSP-2-n11, and S. mitis-CSP-2 + S. mitis-CSP-2-E1Af10r16. Following treatment with S. mitis-CSP-2 or S. mitis-CSP-2-n11, ATCC 49456 was able to internalize spectinomycin resistance, as can be seen by the number of transformants (top and right, respectively). In contrast, ATCC 49456 was unable to internalize spectinomycin resistance following treatment with S. mitis-CSP-2 + S. mitis-CSP-2-E1Af10r16, as determined by the lack of apparent transformants (bottom). The incubation of S. mitis ATCC 49456 with the pFW5-luc plasmid without the addition of synthetic CSP was used as a negative control (left). The experiment was repeated three times in triplicate for a total of nine experiments. See the Supporting information for full experimental details. CSP, competence-stimulating peptide.

Figure 8

Biofilm formation of Streptococcus mitis ATCC 49456 in the presence of S. mitis-CSP-2, S. mitis-CSP-2-n11, S. mitis-CSP-2 together with S. mitis-CSP-2-E1Af10r16 and S. mitis-CSP-2-E1Af10r16 alone.A, biofilm quantification following treatment with the different CSP analogs, as determined via the crystal violet assay. The mean (±S.D.) was as follows: WT, 100% ± 48%; S. mitis-CSP-2, 112% ± 44%; S. mitis-CSP-2-n11, 115% ± 57%; S. mitis-CSP-2 + S. mitis-CSP-2-E1Af10r16, 39% ± 45%; and S. mitis-CSP-2-E1Af10r16, 30% ± 41%. B, representative images of the crystal violet biofilm quantification assay exhibiting the dried biofilms at the bottom of the wells of a 96-well plate (top), the dried biofilms following the crystal violet staining (middle), and the stained wells after the biofilm and crystal violet were dissolved in 30% acetic acid for quantification (bottom). The lead inhibitor, S. mitis-CSP-2-E1Af10r16, with or without exogenous addition of the native S. mitis-CSP-2 signal, was found to decrease the amount of biofilm formed compared to the untreated control. Statistical significance was determined using a one-way ANOVA with Bonferroni’s correction; n.s., not significant; ∗p ≤ 0.1; ∗∗∗p ≤ 0.001. The experiment was repeated three times in triplicate for a total of nine experiments. See the Supporting information for full experimental details. CSP, competence-stimulating peptide.