Abstract
Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune-stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen-associated molecular pattern recognized by extracellular and intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum-specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest antitumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains.
Highlights
Despite the exponential growth of biomedical knowledge over the last decades, we are still facing health conditions that are not controllable
We hypothesized that manipulating the spatiotemporal onset of flagella synthesis or of various steps in flagellar assembly might represent a valid strategy to increase the adjuvant power of Salmonella vector strains without increasing their pathogenicity
Deleting the recombinase Hin, which is responsible for flagellin phase variation 15, as well as removing FljA, the repressor of fliC translation 34, allowed us to influence the composition of the large antigenic flagellar filament: Δhin-5717 (FliC-ON), Δhin-5718 (FljB-ON), ΔfljA Δhin-5718 (FliC-ON & FljB-ON)
Summary
Despite the exponential growth of biomedical knowledge over the last decades, we are still facing health conditions that are not controllable. The impairment of both these two important PAMPs - LPS and flagellum - directly led to a loss of therapeutic potency Such molecules are essential assets of the adjuvanticity of the Salmonella vector. Downregulation to avoid immune recognition by Pattern-Recognition-Receptors (PPR), like extracellular TLR-5 or intracellular caspase-1 is essential for bacterial survival in vivo 12,13 This evasion mechanism already indicates that bacteria constitutively expressing flagella might elicit strong immune activation. In line with these observations, Eom and colleagues observed an enhanced adjuvanticity of Salmonella that co-expressed both flagellin proteins FliC and FljB 17 These results demonstrate that a modulation of synthesis and assembly of flagella might allow to engineer appropriately modified bacterial vector strains for therapeutic applications. We believe that the flagellum of Salmonella represents an ideal target for immunomodulatory modifications and it might be possible to generate optimized safe vector strains with improved adjuvant properties for prophylaxis and therapy
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