Vitamin B3 Triggers Biosynthesis of Secondary Metabolite Dormancy Signals in Streptococcus suis

Abstract

Human-associated streptococci have not been viewed as productive sources of natural products. Against expectation, bioinformatic searches recently revealed a large collection of diverse biosynthetic gene clusters coding for ribosomally synthesized and post-translationally modified peptides (RiPPs) in streptococcal genomes. The most abundant of these, the tqq gene cluster, is specific to Streptococcus suis, a burdensome agricultural pathogen and zoonotic agent. Herein, we used high-throughput elicitor screening to identify both small molecule elicitors and products of the tqq cluster. We show that the B₃ vitamin niacin effectively elicits the tqq cluster leading to the biosynthesis of a family of RiPP natural products, which we termed threoglucins and characterized structurally. The defining feature of threoglucins is an aliphatic ether bond giving rise to a substituted 1,3-oxazinane heterocycle in the peptide backbone. Isolation of 22 congeners of threoglucins facilitated structure activity relationship studies, demonstrating the requirement for the oxazinane substructure and a Trp-Tyr C-terminal dyad for biological activity, namely antibiotic persistence and allolysis at low and high doses, respectively. Potential therapeutic applications of threoglucins are discussed.

Figure 1.

Streptococcal RaS-RiPP network and the tqq cluster. (A) RaS-RiPP network with each node representing a unique RiPP BGC. Subfamilies with known products are labeled. (B) Structures of mature RiPPs from the network. Gray spheres represent unmodified amino acids with the indicated one-letter code. (C) The tqq cluster is adjacent to an shp/rgg QS operon (green) and codes for a precursor peptide (TqqA, sequence shown), a RaS enzyme (TqqB), and a combined peptidase-transporters (TqqC).

Figure 2.

Activation of threoglucin production by HiTES. (A) 3D difference map showing residual features (m/z and abundance) as a function of elicitor. Peaks corresponding to threoglucins are colored blue; structures are shown for the top elicitors. (B, left) 2D slice from the 3D map in panel (A) showing abundance of threoglucin E across all 442 elicitors. Niacin and anabasine-treated samples are indicated. (B, right) Extracted ion chromatograms show relative abundance of threoglucin E from S. suis cultures treated with vehicle-control (Con.), nicotinamide (Nic.), anabasine (Ana.), and niacin (Nia.). (C) Quantification of threoglucins A, B, E, F, I, and O in niacin-treated S. suis cultures as a function of OD₆₀₀.

Figure 3.

Structures of mature threoglucins. (A) Shown are relevant NMR correlations around the threonine-glutamine α-ether cross-link and observed MS/MS b- and y-ion fragments. (B) Structures of 22 threoglucin congeners. Gray spheres represent unmodified amino acids. Curved lines between threonine and glutamine represent the oxazinane heterocycle.

Figure 4.

Biological activity of threoglucin A/B. (A) Growth curve for untreated (black) and threoglucin A/B-treated S. suis with concentrations ranging from 60 nM to 30 μM as shown. (B) Effect of threoglucin A/B addition at OD₆₀₀ of 0.2 on the growth kinetics of S. suis. Traces are color-coded as shown. (C) Survival of S. suis, as measured by CFUs, after treatment of cultures with 30 μM threoglucin A/B at OD₆₀₀ of 0.2. (D) Survival of S. suis after treatment during exponential phase (OD₆₀₀ of 0.2) with 2 μM threoglucin A/B (+Thr.), 2 μM threoglucin A/B and 200 μM ciprofloxacin (+Thr. + Cip.) or 200 μM ciprofloxacin (+Cip.), as measured by CFU analysis. The results are scaled to growth of untreated cultures.