Extended Data Fig. 1: Effects of mutations in Thoeris genes on defence against phage SPO1.
From: Antiviral activity of bacterial TIR domains via immune signalling molecules
a, Plaques of phage SPO1 on control cells (black), cells expressing both WT Thoeris proteins (green), cells expressing mutant ThsA(N112A) and WT ThsB (magenta), or cells expressing WT ThsA and mutant ThsB(E85Q) (cyan). Ten-fold serial dilution of the phage lysate were dropped on the plates. b, Efficiency of plating (EOP) of phage SPO1 on control and Thoeris-containing strains, representing plaque-forming units per millilitre (PFU/ml). Asterisk marks statistically significant reduction in EOP (One-way ANOVA, followed by pairwise multiple comparison analysis according to Tukey’s honest significant difference criterion p=2*10−8). c, Replication of phage SPO1 in the presence of Thoeris-containing and Thoeris-lacking (control) cells, or without cells (no bacteria). Lysates were collected 2.5 h following infection of liquid cultures at an initial MOI of 5, and phage titer was quantified by plating serial dilution of the lysates on the control strain. Asterisk marks statistically significant reduction in EOP compared to the control strain (One-way ANOVA, followed by pairwise multiple comparison analysis according to Tukey’s honest significant difference criterion p=0.016). d, Remaining phage titre after culture recovery. Samples were collected 12 h following infection of liquid cultures at an initial MOI of 5, and phage titre was quantified by plating serial dilution of the samples on the control strain. Asterisk marks statistically significant reduction in EOP compared to the control strain (One-way ANOVA, followed by pairwise multiple comparison analysis according to Tukey’s honest significant difference criterion p = 0.0004). e, Growth curves of infections with phage SPO1 at MOI of 5 of control cells (black), Thoeris-expressing cells (dark green), and 11 colonies isolated from recovered Thoeris-expressing cells 12 h post infection (light green). 3–4 Colonies were isolated from each of three independent infections. f, g, Growth curves of uninfected Thoeris mutants (f) and during infection by phage SPO1 at MOI of 5 or 0.05 (g). Three independent experiments are presented as individual curves. h, Adenosine diphosphate ribose (ADPR) levels in control culture (black) and cells expressing wild type Thoeris (green). Time 0 represents uninfected cells. Cells were infected by phage SPO1 at an MOI of 5. Each line represents the mean of three independent experiments, with individual data points shown. ADPR levels were measured by LC–MS and calculated from the area under the curve of the identified ADPR peak. i, NAD+ levels in cell expressing WT ThsB + ThsA(N112A) (magenta) and cells expressing WT ThsA + ThsB(E85Q) (cyan). Time 0 represents uninfected cells. Each line represents the mean of three independent experiments, with individual data points shown. Cells were infected by phage SPO1 at an MOI of 5. NAD+ levels were measured by LC–MS and calculated from the area under the curve of the identified NAD+ peak.
