Fig. 4: Combining defense systems enhances phage resistance.

A CR1-immune S. thermophilus DGCC7710 was transformed with the pTRKL2 vector carrying either Gabija, Hachiman, or Thoeris. Strains expressing combinations of two defense systems were compared to strains carrying individual systems. Spot tests illustrate resistance profiles against phage D5691 (encoding AcrIIA6, which inhibits CR1) and phage 2972 (lacking known ACR). B Liquid culture assays comparing CR1 + Gabija combinations against phages 2972 and D5691 at initial MOIs ranging from 0.0005 to 50. Control strains include wild-type DGCC7710 (WT) and CR1-immune DGCC7710 (CR1BIM) carrying the empty pTRKL2 vector. The area under the curve (AUC) was calculated for each condition, and the relative AUC was determined by normalizing to the non-infected control. Synergy between defense systems is indicated by a black asterisk, and additive effects are indicated by a red asterisk. C Relative AUC values for combinations of CR1 with either Thoeris or Hachiman against phages 2972 and D5691. The term “System” refers to Thoeris or Hachiman as indicated. Synergy and additive effects are indicated by an asterisk color in black and red, respectively. D Quantification of escape phages recovered after liquid assays at MOI 0.05 with phages 2972 and D5691 for each individual and combined defense system. Filled circles indicate the presence of countable plaques, while hollow red circles signify lysis, but plaques were not counted. E Schematic illustration of pyramiding (combining two defense systems in the same strain) versus mixing (co-culture of two strains, each expressing a different system). F Comparison of pyramiding versus mixing strategies for three combinations: CR1 + Gabija, Dodola + Gabija, and CR1 + Thoeris. For all experiments, lines or bars represent the mean value of biological replicates (n = 3), with shaded areas indicating confidence intervals and error bars indicating standard deviation. The statistical significance of the synergy scores was evaluated using a two-sided one-sample t test with Benjamini-Hochberg correction for multiple testing. A positive score significantly different from zero (p-value < 0.05) indicated synergy, while a score not significantly different from zero indicated an additive effect.