Fig. 5. Toxin production and release in vitro.

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Fig. 5. Toxin production and release in vitro. Toxin production and release in vitro. (A) In vitro cell lysate and culture supernatant samples from R20291, FM2.5, and FM2.5RW cultures were normalized to an equivalent optical density and separated on 6% SDS polyacrylamide gels. Toxin B was detected by Western immunoblot using an anti–toxin B monoclonal antibody. Samples were taken at the indicated time points. (B) To determine whether Avidocin-CD killing released intracellular toxin, R20291 was incubated for an hour either with Av-CD291.2 at the indicated ratio of agent to cells or Av-CD684.1, which does not kill strain R20291, at a 500:1 ratio (IS), or left untreated (UT). After treatment, viable bacteria were enumerated (bar graph), and the percentage killed relative to the untreated control was determined (numbers above each bar). The number of spores present in the untreated sample was determined after heat treatment at 65°C for 30 min to kill vegetative cells (HT). (C) After Avidocin-CD treatment, released toxin B in culture supernatants (ExC) was detected by Western immunoblot using an anti–toxin B monoclonal antibody. As a positive control for toxin release, R20291 was treated with the phiCD27L bacteriophage endolysin (41). The amount of remaining intracellular toxin B (IntC) was determined by lysing cells with CD27L endolysin and detection by Western immunoblot as before. A fresh sample of untreated R20291 was lysed with CD27L endolysin to show normal intracellular toxin quantities (EL). The original uncropped images for each Western immunoblot can be found in fig. S9. Joseph A. Kirk et al., Sci Transl Med 2017;9:eaah6813 Published by AAAS