Suppression of Salmonella Effectors with CRISPRi Controls the Immune Response to Bacterial Therapies

Many bacterial immunotherapies, because of their intracellular lifestyle, have the potential to overcome the limitations that reduce the efficacy of immune checkpoint inhibitors. One reason this approach is not completely curative is the innate immune suppression of the bacteria. After cell invasion, Salmonella injects effector proteins that inhibit host cell signaling and suppress subsequent immune responses. To increase the efficacy of bacterial therapies we designed a CRISPRi (clustered regularly interspaced short palindromic repeats─interference) system to repress the expression of two Salmonella effectors, SopB and SteE. We hypothesized that repressing SopB decreases AKT phosphorylation and repressing SteE decreases STAT3 phosphorylation. For each effector, we optimized the response by creating multiple guide RNAs and evaluating their ability to repress expression of fluorescent fusion proteins. To characterize their biological effects, we administered these engineered bacteria to cancer cells and macrophages. In cancer cells, CRISPRi repression of SopB reduced activation of AKT and increased cellular apoptosis. In both cancer cells and macrophages, repression of SteE reduced activation of STAT3, reduced the secretion of immunosuppressive IL-10 and increased the secretion of pro-inflammatory TNF-α. Promotion of apoptosis and secretion of IL-10 are biological functions downstream of AKT and STAT3, respectively. The increased production of TNF-α was most likely mediated by the reduced levels of IL-10. These experiments show, for the first time, that CRISPRi modulation of effector expression in therapeutic Salmonella can control the physiology of mammalian cells and shift their phenotype to one that is less immunosuppressive and more favorable for cancer therapy.