A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision

Abstract

Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA (VqmA_Vc), which together repress genes for virulence and biofilm formation. vqmA genes exist in Vibrio and in one vibriophage, VP882. Phage-encoded VqmA (VqmA_Phage) binds to host-produced DPO, launching the phage lysis program via an antirepressor that inactivates the phage repressor by sequestration. The antirepressor interferes with repressors from related phages. Like phage VP882, these phages encode DNA-binding proteins and partner antirepressors, suggesting that they, too, integrate host-derived information into their lysis-lysogeny decisions. VqmA_Phage activates the host VqmA_Vc regulon, whereas VqmA_Vc cannot induce phage-mediated lysis, suggesting an asymmetry whereby the phage influences host QS while enacting its own lytic-lysogeny program without interference. We reprogram phages to activate lysis in response to user-defined cues. Our work shows that a phage, causing bacterial infections, and V. cholerae, causing human infections, rely on the same signal molecule for pathogenesis.

Keywords: DPO; Qtip; Vibrio; VqmA; antirepressor; autoinducer; kill switch; lysis-lysogeny; phage; quorum sensing.

Figure 1:. Phage VP882 encodes a homolog of vibrio VqmA that binds DPO and promotes host cell lysis.

(A) Organization of a region of the phage VP882 genome. Colors denote genes characterized here. telN and repA are conserved across linear plasmid-like phage. gp56 (purple) encodes the VqmA_Phage QS receptor, gp55 (red) encodes the Qtip antirepressor, gp59 (green) encodes the cI repressor, gp62 (blue) encodes the Q antiterminator, and gp69-71 (yellow) encode the lysis genes. This color coding is used throughout this work. (B) Growth curves of V. parahaemolyticus lysogenized with phage VP882 (diamonds) or cured of phage VP882 (circles) harboring inducible vqmA_Phage. As indicated, vqmA_Phage expression was induced with 0.2% ara. (C) Growth curves of V. parahaemolyticus lysogenized with phage VP882 harboring inducible vqmA_Phage in minimal medium lacking threonine. As indicated, vqmA_Phage expression was induced with 0.2% ara and DPO was present at 10 μM. (D) Quantitation of DPO from purified proteins in a DPO-dependent E. coli reporter assay. The left bar shows the 10 μM synthetic DPO standard. RLU denotes relative light units. (E) Quantitation of DPO from the extracts in panel D by LC-MS. The left bar shows the 100 μM synthetic DPO standard. AU denotes arbitrary units. Data are represented as mean ± std with n=3 biological replicates (B, D), n=3 technical replicates (E), and n=3 biological and n=3 technical replicates (C). See also Figures S1 and S2.

Figure 2:. Gp62 acts downstream of VqmA_Phage and is required for lysis.

(A) Lysis of V. parahaemolyticus lysogenized with VP882, VP882 gp62::Tn5, and VP882 with a Tn5 insertion downstream of gp62 (control Tn5). See key for introduced vectors/genes. (B) Lysis of V. parahaemolyticus lysogenized with VP882 vqmA_Phage::Tn5 or VP882 gp62::Tn5. See key for introduced vectors/genes. Phages were transmitted to host strains by conjugation (see Methods). Where indicated, 50 ng mL⁻¹ MMC was added. In both panels, all strains were grown in medium containing 0.2% arabinose. Data are represented as mean ± std with n=3 biological replicates.

Figure 3:. Gp62 is an antiterminator and Gp59 is a repressor of the gp69-71 operon encoding the phage lysin genes.

(A) Growth of E. coli carrying arabinose inducible gp69-71 on a plasmid in medium lacking (black) or containing (white) 0.4% arabinose. (B) E. coli carrying a plasmid with Pgp69-lux and a second empty plasmid or the plasmid with arabinose inducible gp62. Black; no arabinose, white 0.2% arabinose. (C) Pq-lux expression from V. parahaemolyticus harboring phage VP882 (denoted Lysogen), lacking VP882 (denoted Phage Cured), or lacking VP882 with gp59 expressed from a plasmid (denoted Phage Cured gp59). (D) Pq-lux expression from E. coli carrying an empty plasmid or the plasmid with arabinose inducible gp59. Black; no arabinose, white; 0.2% arabinose. (E) Western blot analysis of lysate from recA⁺ and ΔrecA E. coli carrying HIS-HALO-Gp59. Symbols: −; no MMC, +; 250 ng mL⁻¹ MMC, M; Marker (PageRuler Plus; representative bands are labeled). (F) Pq-lux expression from the strains in panel E. Black; no MMC, white; 250 ng mL⁻¹ MMC. (G) Time-course of Pq-lux (closed circles) and Pgp69-lux (open circles) production from V. parahaemolyticus lysogenized with phage VP882 following 50 ng mL⁻¹ MMC treatment (top panel) and from V. parahaemolyticus lysogenized with phage VP882 and carrying arabinose inducible vqmA_Phage following addition of 0.2% arabinose (bottom panel). Data are represented as mean ± std with n=3 biological replicates (A, B, C, D, F), and mean ± std with n=3 biological and n=3 technical replicates (G). See also Figure S3.

Figure 4:. VqmA_Phage activates production of Qtip, a phage-encoded small protein that inactivates the cI repressor by sequestration.

(A) E. coli carrying a plasmid harboring Pq-lux, cI, and arabinose inducible vqmA_Phage and a second empty vector, the vector containing a phage genomic fragment encoding gp55 (denoted Active Fragment), or the cloned gp55 gene under the tetA promoter. Where indicated, arabinose and aTc were provided at final concentrations of 0.2% and 100 ng mL⁻¹, respectively. (B) Growth of V. parahaemolyticus harboring phage VP882 and a vector control, the vector with arabinose inducible vqmA_Phage, or the vector with aTc inducible gp55. Arabinose, aTc, and MMC were added at 0.2%, 10 ng mL⁻¹, and 50 ng mL⁻¹ respectively. (C) SDS-PAGE analysis of proteins isolated from the following: Sample 1: cells producing HIS-Gp55 alone. Sample 2: cells producing HIS-Gp55 combined with cells producing the HALO-tag (not fused to cI). Sample 3: cells producing HIS-Gp55 combined with cells producing HALO-cI. Sample 4: cells producing HALO-cI in the absence of HIS-Gp55. Proteins were isolated by applying the indicated lysates to cobalt beads, which bind the HIS-tag fused to Gp55. Thus, proteins interacting with HIS-Gp55 are retained on the beads. Lysates (Input), wash (Wash), and protein remaining on cobalt beads (Beads). The gel was stained for HALO using HALO-TMR. Locations of HALO-cI and the HALO-tag are marked with black and white arrowheads, respectively. No other obvious protein bands were present in the bead sample containing HALO-cI and HIS-Gp55 suggesting that Gp55 is specific for cI. Marker (M, PageRuler Plus; representative bands are labeled). (D) Confocal microscopy of E. coli producing HIS-HALO-cI in the absence of Qtip (top left), HIS-HALO-cI combined with 100 ng mL⁻¹ aTc induction of qtip (top right), HIS-HALO (not fused to cI) combined with 100 ng mL⁻¹ induction of qtip (bottom left), HIS-HALO-cI combined with 250 ng mL⁻¹ MMC (bottom right). (E) Confocal microscopy of E. coli producing HIS-HALO-cI and SNAP-Qtip. HALO-cI was visualized with HALO-TMR and SNAP with JF₅₀₃. The medium contained 100 ng mL⁻¹ aTc. (F) Confocal microscopy of E. coli producing HALO-tagged cI_VP882, cI_MJ1 or cI_lambda (top row), co-producing Qtip (middle row) or co-producing ORF₅₈₄ (bottom row). Qtip and ORF₅₈₄ production were induced with 100 ng mL⁻¹ aTc. (G) Growth of V. parahaemolyticus carrying phage VP882 and aTc inducible qtip or orf₅₈₄. Black; no aTc, white; 10 ng mL⁻¹ aTc. All scale bars are 3 pm. Data in A, B, and G are represented as mean ± std with n=3 biological replicates. See also Figures S4 and S5.

Figure 5:. Asymmetric gene regulation by VqmA_Phage and VqmA_Vc

(A) Fluorescence from PvqmR-mKate2 in ΔvqmA V. cholerae carrying a vector control, vqmA_Vc under its native promoter, or arabinose inducible vqmA_Phage. The medium contained 0.2% arabinose. RFU denotes relative mKate2 fluoresence units. (B) Electrophoretic mobility shift assay with PvqmR DNA (left, white arrowheads) and Pqtip DNA (right, black arrowheads) together with VqmA_Vc, VqmA_Phage, or no protein (denoted -). Proteins used at 130 nM and 16.25 nM (denoted 8x and 1x, respectively). DNA probes = 540 pM. (C) E. coli carrying a plasmid with Pqtip-lux (black) or PvqmR-lux (white) and a second plasmid with either arabinose inducible vqmA_Phage or vqmA_Vc. Medium contained 0.2% arabinose. We estimate that VqmA_Vc binds to its own (PvqmR) promoter 456-fold better than the non-cognate promoter (Pqtip), while VqmA_Phage binds its target promoter (Pqtip) 4.5-fold better than the non-cognate promoter (PvqmR). PvqmR is activated 7-fold more strongly by the cognate VqmA (VqmA_Vc) than by the non-cognate VqmA_Phage. In contrast, Pqtip is activated 278-fold more strongly by the cognate VqmA (VqmA_Phage) than by the non-cognate VqmA_Vc. See Methods for details on quantitative comparisons. (D) Growth of ΔvqmA V. cholerae lysogenized with phage VP882 and either a vector carrying vqmA_Vc (left set of bars) or vqmA_Phage (right set of bars). Black; no arabinose, white; 0.2% arabinose. In A, C, and D, data are represented as mean ± std with n=3 biological replicates. See also Figure S6.

Figure 6:. Quorum-sensing controls the lysis-lysogeny fate decision in phage VP882.

(A) Phage VP882 (multi-colored ring) can lyse or lysogenize its vibrio host. In the lysogenic state, Q (blue) production is repressed by cI (green). Lysis depends on inactivation of cI activity, and that is mediated by two independent inputs, host DNA damage or QS. Host DNA damage (lightning bolt) leads to RecA-assisted proteolysis (scissors) of the cI repressor. The QS input is mediated by VqmA_Phage (purple) binding to the host-produced DPO AI, which is derived from threonine via the Tdh enzyme. VqmA_Phage bound to DPO activates expression of qtip (red). Qtip aggreagates the cI protein. Irrespective of the input, reduced cI activity leads to derepression of q and subsequent expression of genes involved in lysis (yellow). VqmA_Phage, when bound to host DPO, also activates transcription of the host VqmA QS target, vqmR, leading to production of the sRNA VqmR. The VqmR regulon includes genes required for biofilm formation. (B) Growth of WT and Δtdh V. cholerae lysogens. Left; carrying inducible vqmA_Phage on a plasmid. Right; carrying inducible qtip on a plasmid. Top; no addition. Bottom; with 100 μM DPO. All strains carry phage VP882 vqmA_Phage::Tn₅. When present, as indicated in the associated keys, the inducers of vqmA_Phage and qtip (arabinose and aTc, respectively), were present at 0.035% and 10 ng mL⁻¹, respectively. (C) qPCR of phage DNA prepared from the samples in (B). Black; Δtdh, white; WT V. cholerae. Viral Load is the amount of VP882-specific DNA in the induced samples relative to the uninduced samples, in all cases relative to DNA of a non-phage plasmid (see Methods). Data are represented as mean ± std with n=3 biological replicates (B) and as mean ± sem with n=3 biological replicates and n=4 technical replicates (C). See also Figures S6 and S7.

Figure 7:. Reprogrammed phage as kill switches.

(A) A V. cholerae-specific phage kill switch. Phage VP882 q::Tn5 (depicted as the multi-colored ring lacking the blue q gene) exists as a lysogen that is unable to lyse host cells. A functional copy of q, under a V. cholerae-specific promoter (PvqmR-q, depicted as the plasmid carrying the blue gene) is not activated in E. coli (left) because E. coli lacks VqmA, but is activated in V. cholerae that possesses VqmA (middle, blue waves showing Q production and yellow waves showing lysis,). ΔvqmA V. cholerae, by contrast does not activate q (right). We note that all three strains produce DPO. The key for this kill switch is the selective presence of VqmA_Vc only in the WT V. cholerae host. (B) Killing of WT V. cholerae and growth of ΔvqmA V. cholerae, WT V. parahaemolyticus, and WT V. vulnificus using the V. cholerae-specific targeting strategy from panel A. Relative colonies recovered is the number of exconjugant colonies obtained after the plasmid carrying PvqmR-q was introduced into the indicated strains compared to when a control plasmid was introduced. All strains carry phage VP882 q::Tn5 as a lysogen. (C) S. typhimurium-specific kill switch. Growth of S. typhimurium carrying a plasmid with aTc inducible hilD, the Pinvf-q kill switch, and phage VP882 harboring q::Tn5 (left two bars), inducible hilD and the Pinvf-q kill switch without phage VP882 q::Tn5 (middle two bars), inducible hilD and phage VP882 q::Tn5 without the PinvF-q kill switch (right two bars). Black; no aTc, white; 2 ng mL⁻¹ aTc. In B and C, data are represented as mean ± std with n=3 biological replicates. See also Figure S6.