Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

Electronic AND gates are created by placing two transistors in series; similarly, we sequentially dosed bacteria with two prodrugs, each with corresponding BAH values to represent the gate inputs (i.e., A and B). We controlled the corresponding BAH value for each prodrug with the linker 25 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint substrate sequence (e.g., A or B = 0 when kcat = 7 x 10 7 s -1 , A or B = 1 when kcat = 2 x 10 6 s -1 ) and tested all four combinations of the two prodrugs (i.e., AB = 00, 01, 10, and 11). When exposed to all four possible inputs, bacteria only survived the condition with two low kcat prodrugs (i.e., AB = 11), which matched the ideal outputs of an AND gate (Fig. 5B) . These results show that dosing multiple prodrugs in sequence reduces the fraction of bacterial populations that survive (e.g., one 5 prodrug = 50% survival, two prodrugs = 25% survival, etc.). Whereas the AND gate comprised prodrugs in series, we demonstrated one implementation of an OR gate by splitting a population of bacteria in half (i.e., separate wells), dosing each with a different prodrug during the same time interval (i.e., in parallel) and recombining the bacteria post-treatment. Using this circuit, bacteria survived in any case where at least one half of the population was dosed with low kcat prodrug 10 (i.e., AB = 01, 10, or 11), matching the ideal outputs of an OR gate (Fig. 5C) . We created a NOT gate by using a P-type transistor comprising ampicillin-loaded heat-triggered liposomes, which caused bacteria to die at high temperatures (i.e., IN = 1) and survive at low temperatures (i.e., IN = 0). (Fig. 5D) . These results show that heat-triggered liposomes can be used as a fail-safe to kill bacteria above the critical temperature representing the onset of the defiance phenotype. To 15 validate the multi-prodrug circuit, we incubated populations of bacteria under each of the eight environmental conditions with three prodrugs: (1) an AMP prodrug with fixed kcat/Km, (2) an AMP prodrug with kcat/Km determined by input C, and (3) a heat-triggered, drug-loaded liposome (Fig. 4A) . Whereas a single prodrug only eliminated bacteria in half of the input cases (Fig. 5E) , our circuit autonomously eliminated bacteria under all eight combinations of the three environmental 20 signals (Fig. 5F) . These results demonstrate that by utilizing the transistor-like properties of prodrugs, we can design and administer biocircuits that combat bacterial defiance. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint environmental conditions. (E) Bacterial viability assay demonstrating the eight outputs of the autonomous circuit. (F) Representative CFU images for single prodrug and multi-prodrug treatment (scale bar = 4 mm). Error bars represent standard deviation (n = 3). * < 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001.