Streptococcus pneumoniae detects and responds to foreign bacterial peptide fragments in its environment

Abstract

Streptococcus pneumoniae is an important cause of bacterial meningitis and pneumonia but usually colonizes the human nasopharynx harmlessly. As this niche is simultaneously populated by other bacterial species, we looked for a role and pathway of communication between pneumococci and other species. This paper shows that two proteins of non-encapsulated S. pneumoniae, AliB-like ORF 1 and ORF 2, bind specifically to peptides matching other species resulting in changes in the pneumococci. AliB-like ORF 1 binds specifically peptide SETTFGRDFN, matching 50S ribosomal subunit protein L4 of Enterobacteriaceae, and facilitates upregulation of competence for genetic transformation. AliB-like ORF 2 binds specifically peptides containing sequence FPPQS, matching proteins of Prevotella species common in healthy human nasopharyngeal microbiota. We found that AliB-like ORF 2 mediates the early phase of nasopharyngeal colonization in vivo. The ability of S. pneumoniae to bind and respond to peptides of other bacterial species occupying the same host niche may play a key role in adaptation to its environment and in interspecies communication. These findings reveal a completely new concept of pneumococcal interspecies communication which may have implications for communication between other bacterial species and for future interventional therapeutics.

Keywords: Streptococcus pneumoniae, bacteria; interspecies communication; non-encapsulated; peptide.

Figure 1.

Identification of AliB-like ORF 1 ligand SETTFGRDFN and AliB-like ORF 2 ligands FPPQSV and FPPQS. (a) AliB-like ORF 1 ligand: extracted ion chromatogram of the doubly charged peptide ion with mass-to-charge ratio (m/z) of 587.26201 (retention time (RT) 47.36 min) with mass tolerance of ±1 ppm from the negative control (i) and positive sample (ii), intensity scale set at the same level. (b) Representative fragment mass spectrum acquired in the linear trap quadrupole (LTQ) iontrap and sequence interpretation by Phenyx software. Only the y-ion series (fragments appearing to extend from the carboxyl terminus) is annotated. (Individual peaks corresponding to the initial two amino acids S and E at the amino terminus are not visible in the y-ion series and are therefore not annotated but SE is the mass difference between the last detectable y-ion at 957.59 and the singly charged molecular ion of 1173.517.) (c) AliB-like ORF 2 ligands: extracted ion chromatogram of the singly charged peptide ions with mass-to-charge ratio (m/z) of 575.27934 (RT = 45.45 min, corresponding to sequence FPPQS) and 674.34844 (RT = 51.05 min, sequence FPPQSV) with mass tolerance of ±2 ppm from the negative control (i) and positive sample (ii), intensity scale set at the same level. A representative fragment mass spectrum acquired in Fourier transformation (FT) mode, resolution of 7500, of peptides 575 (d) and 674 (e) is shown. Manual interpretation of the fragment peaks is shown using italic letters for the b-ion (fragments appearing to extend from the amino terminus) and non-italic letters for the y-ion series. Confirmation of correct interpretation was achieved with fragment spectra of synthetic peptides (electronic supplementary material, figure S3).

Figure 2.

Binding of recombinant AliB-like ORFs to their ligands. Binding of recombinant AliB-like ORF 1 (a–d) and AliB-like ORF 2 (e–k) to synthetic peptides was determined by measuring changes in intrinsic protein fluorescence using tryptophan fluorescence. A drop in fluorescence indicates binding. Peptide sequences are indicated above the arrows marking the moment of their addition to the protein.

Figure 3.

Competence gene expression and transformation. (a) Percentage transformation rates of strain 110.58 and its mutants ΔORF 1 + 2, ΔORF 1 and ΔORF 2, showing the mean of three independent experiments. Error bars show s.e.m. *p = 0.0058, **p = 0.0022, ***p < 0.0001. (b) The synergistic effect of pre-incubation with 100 µg ml⁻¹ AliB-like ORF 1 ligand (SETTFGRDFN) on CSP 2-mediated competence induction was determined by measuring the expression of late competence gene ssbB using a transcriptional fusion of the ssbB promoter region with the luciferase gene, displayed as AUC of the luminescence signal. Results represent three experiments and show s.e.m. For each strain, the expression of ssbB in the presence of ORF 1 ligand is presented as a fold difference compared with the expression value of that strain in the absence of the ORF 1 ligand. *p = 0.0311.

Figure 4.

Effect of incubation with E. coli on pneumococcal growth. The wild-type strain 110.58 and its mutants, as well as the control laboratory strain R6, were assessed for growth in the presence and absence of E. coli bacteria by quantifying colony forming units (cfu). Data represent the mean of three independent experiments. Error bars show s.e.m. *p < 0.05.

Figure 5.

Adherence to Detroit 562 nasopharyngeal epithelial cells. The number of adhered bacteria per Detroit cell was counted by microscopy following Giemsa staining. Data represent the mean of four independent experiments. Error bars show s.e.m.

Figure 6.

Colonization of the nasopharynx of female MF1 mice. Quantification of bacterial load in the nasopharynx at different timepoints post-inoculation was determined by plating out dilutions of nasopharyngeal tissue homogenate and counting the number of colony-forming units for the wild-type strain 110.58 (solid lines with circles), its mutant lacking the aliB-like ORFs (ΔORF 1 ± 2) (dashed lines with squares), ΔORF 1 (solid line with triangles) and ΔORF 2 (dotted lines with inverted triangles). All data derive from five mice per timepoint per strain tested. Data are expressed as mean log₁₀ cfu/ml nasopharyngeal tissue ± s.e.m.