Peptide maturation molecules act as molecular gatekeepers to coordinate cell-cell communication in Streptococcus pneumoniae

Abstract

The human pathogen Streptococcus pneumoniae (Spn) encodes several cell-cell communication systems, notably multiple members of the Rgg/SHP and the Tpr/Phr families. Until now, members of these diverse communication systems were thought to work independently. Our study reveals that the ABC transporter PptAB and the transmembrane enzyme Eep act as a molecular link between Rgg/SHP and TprA/PhrA systems. We demonstrate that PptAB/Eep activates the Rgg/SHP systems and represses the TprA/PhrA system. Specifically, they regulate the respective precursor peptides (SHP and PhrA) before these leave the cell. This dual mode of action leads to temporal coordination of these systems, producing an overlap between their respective regulons during host cell infection. Thus, we have identified a single molecular mechanism that targets diverse cell-cell communication systems in Spn. Moreover, these molecular components are encoded by many gram-positive bacteria, suggesting that this mechanism may be broadly conserved.

Keywords: CP: Microbiology; Eep; Gram-positive; PptAB; RRNPPA; Rgg/SHP; Streptococcus pneumoniae; TprA/PhrA; cell-cell communication; colonization; quorum sensing.

Figure 1.. The transporters PptAB and AmiA-F and the protease Eep are required for activation of Rgg/SHP systems in Spn strain D39

(A) Schematic displaying SHP processing and signaling for selected Rgg/SHP systems in Spn strain D39* (*Rgg/SHP939 is not active in the conditions tested, Figures S1B, S1E, and S1F), which has been described in other streptococcal species.^, The model displays the precursor SHP being exported via the transporter PptAB and processed via the membrane protease Eep. The processed SHP enters the cells via the transporter AmiA-F and binds its cognate Rgg regulator to activate gene expression of adjacent genes and in the genome (indicated by “+”). Gene sizes are not to scale. Image created with BioRender.com. (B) WT and peptide maturation molecule mutants (ΔpptAB, Δeep, ΔamiA-F) were grown in CDM-glucose (55 mM) until early log phase (OD₆₀₀ = 0.3), and gene expression of target genes that are regulated by the described Rgg/SHP systems was measured via RT-qPCR. Gene expression is plotted relative to WT. (C) Plot of Pshp144:lux reporter over time in diverse backgrounds for cells grown in CDM-glucose (55 mM) in the presence of 1 μM final concentration (f.c.) synthetic SHP144-C12 peptide (EWVIVIPFLTNL). (D) To indirectly quantify SHP144 export, we used the Pshp144:lux reporter strain lacking endogenous shp144. The WT, ΔpptAB, or ΔamiA-F strains were grown in CDM-glucose (55 mM) and mixed with the reporter at a 1 to 1 ratio, and activity of the reporter was measured at the indicated time points. All experiments were carried out in triplicate, graphs depict values with standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA for multiple comparisons (*p < 0.05, ****p < 0.0001; ns, not significant).

Figure 2.. The Spn TprA/PhrA locus is negatively regulated by PptAB and Eep in an Rgg/SHP-system-independent manner

(A) Schematic displaying PhrA peptide processing and signaling in Spn strain D39. The precursor form is predicted to be exported via the Sec pathway. The processed form is then imported via AmiA-F into producing or neighboring cells, where it binds TprA, its cognate regulator, releasing repression and activating expression of genes in the adjacent locus (predicted lantibiotic gene cluster) and elsewhere in the genome. Gene size is not to scale. Image created with BioRender.com. (B) PphrA:lux reporter in WT and mutant backgrounds was grown in CDM-glucose (55 mM), and bioluminescence was quantified at indicated optical densities. (C) WT and mutants (ΔtprA, ΔpptAB, Δeep, Δrgg/shp_all, and Δrgg/shp_allΔpptAB) were grown in CDM-glucose (55 mM) until early log phase (OD₆₀₀ = 0.3), and gene expression of the regulator tprA, the signaling peptide phrA, and the target gene lanM was measured via RT-qPCR. Gene expression is plotted relative to WT (dotted line). (D) PphrA:lux double-deletion mutant reporter strains were grown in CDM-glucose (55 mM), and bioluminescence was quantified at the indicated optical densities. Dotted line indicates highest bioluminescence levels for ΔpptAB. (E) Schematic showing inhibition of TprA/PhrA, but not TprB/PhrB or TprC/PhrC, via PptAB that is independent of the Rgg/SHP144 system in Spn strain TIGR4. (F) TIGR4 WT and mutants were grown in CDM-glucose (55 mM) until early log phase (OD₆₀₀ = 0.3), and gene expression of tpr regulators and phr peptides was quantified via RT-qPCR relative to WT. All experiments were carried out in triplicate, graphs depict values with standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA for multiple comparisons (**p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3.. PptAB negatively regulates the PhrA precursor peptide

(A) To indirectly quantify PhrA secretion, we used the PphrA:lux reporter strain lacking endogenousphrA. The WT, ΔtprA, or ΔpptAB strains were grown in CDM-glucose (55 mM) and mixed with the reporter at a 1 to 1 ratio, and activity of the reporter was measured at the indicated time points. (B) Strains containing the PphrA:lux reporter were grown in CDM-glucose (55 mM), and promoter activity was measured at the indicated optical densities. Dotted line indicates the highest bioluminescent signal in the ΔpptAB strain. (C) Schematic displaying PptAB-mediated repression of precursor PhrA (created with BioRender.com). (D) Representative confocal microscopy images of WT and mutant strains that were incubated with 1 μM synthetic PhrA-C10 that has an N-terminally conjugated Alexa 546 fluorophore. Images were taken at 100× original magnification and processed with ImageJ as described in the STAR Methods. Scale bar, 10 μm. (E) Quantification of phrA promoter activity as measured with the PphrA:lux reporter in WT (i), ΔpptAB (ii), ΔamiA-F (iii), and ΔpptABΔamiA-F (iv) incubated with 1 μM synthetic PhrA-C10 (blue line) or _{Alexa 546}PhrA-C10 (red line). Measurements were taken at indicated time points and compared to DMSO control (black line). All experiments were carried out in triplicate, graphs depict values with standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA for multiple comparisons (*p < 0.05, ****p < 0.0001).

Figure 4.. PptAB coordinates Spn cell-cell communication systems in the presence of host cells

(A–F) WT, ΔpptAB, ΔamiA-F, and WT + synthetic peptides (cocktail of 1 μM each PhrA-C10, RtgS1-C10, SHP144-C12, and SHP1518-C12) cells were incubated with A549 lung epithelial cells (MOI 1:100), and gene expression changes of relevant genes were measured. Fold changes are plotted relative to the WT 0 h time point, and mutant/treated WT were compared to WT at individual time points. (G) Bacterial counts at indicated time points measured as colony forming units (CFU) per milliliter. (H and I) Bioluminescent peptide reporters lacking endogenous peptide were treated with cell-free supernatants from WT, ΔpptAB, ΔamiA-F, or peptide-deletion mutants harvested at multiple time points during A549 cell infection. Final time point for PhrA activity is displayed at 3.5 h post treatment (H) and final time point for SHP144 is displayed at 1 h post treatment (I). All experiments were carried out in triplicate, graphs depict values with standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 5.. The N-terminal domain of PhrA mediates its secretion as well as its interaction with PptAB

(A) Alignment of D39 signaling peptides PhrA (and respective mutants), RtgS1, SHP144 (and mutant), SHP939, and SHP1518, highlighting conserved N-terminal positively charged residues (red) as well as a hydrophobic stretch in PhrA (green) and C-terminal functional domain (orange). (B) Bacterial counts at indicated time points measured as colony forming units (CFU) per milliliter. (C–F) PhrA_RKKR::AAAA, PhrA_RKKR::AAAAΔpptAB, PhrA_Δaa10-21, PhrA_Δaa2-46, and SHP144_KKRK::AAAA cells were incubated with A549 epithelial cells (MOI 1:100), and gene expression changes of tprA, phrA (except PhrA_Δaa2-46, sequence too short), lanM, and vpoB1 were measured. Fold changes are plotted relative to the WT 0 h time point, and mutants were compared to WT (replotted from Figure 4) at individual time points. (G and H) Bioluminescent reporters for secreted PhrA at 3.5 h post treatment (G) and SHP144 at 1 h post treatment (H) were treated with cell-free supernatants of respective mutants from selected time points of A549 infection. Dotted lines indicate maximum levels from supernatants of cells lacking endogenous peptide. All experiments were carried out in triplicate, graphs depict values with standard error of the mean (SEM), and statistical analysis was performed using two-way ANOVA for multiple comparisons (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6.. PptAB is a virulence factor that coordinates Spn cell-cell communication signals in vivo

(A and B) Mice (n = 10 per group) were infected with 4 × 10⁶ CFU of WT, ΔphrA, ΔpptAB, or ΔpptABΔphrA using the pneumonia model. Bacterial counts were determined in the blood at 16 and 24 h post infection. Individual data points and median are plotted. Differences in CFU counts were calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ****p < 0.0001). (C and D) Utilizing the pneumonia mouse model of infection, P_phrA::CBRluc (C) and P_shp144::CBRluc (D) reporters in WT and ΔpptAB backgrounds were imaged at 2 h post infection as shown on the right (n = 10 animals per group, except for the mouse marked with “+” in (D), which was excluded due to lack of signal). Quantifications on the left were carried out using luminescence over lung normalized to CFU/mg of lung tissue. Differences between strains were determined using the Mann-Whitney test (*p < 0.05).