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Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation and damage in which fibroblast-like synoviocytes (FLS) play a central role. Invasiveness and proliferation of FLS in RA is dependent on activity of the K Ca 1.1 potassium channel. Peptide blockers of K Ca 1.1, such as iberiotoxin (IbTX), can be delivered subcutaneously to treat animal models of RA. We tested whether an engineered probiotic oral delivery platform could effectively deliver IbTX systemically in a rat model of RA. Results A plasmid for inducible secretion of IbTX was constructed and transformed into probiotic Limosilactobacillus reuteri ATCC PTA-6475 to generate LrIbTX. No differences in growth rate between LrIbTX and the control strain were detected in vitro, and live LrIbTX was recovered from the feces of rats following oral gavage. IbTX was detected in the sera of healthy rats orally gavaged with LrIbTX by a K Ca 1.1 competitive binding assay using a biotinylated IbTX analog and streptavidin-conjugated fluorophore. Collagen-induced arthritis (CIA), an animal model of RA, was used to measure the effect of LrIbTX versus injected IbTX or control L. reuteri expressing an irrelevant protein on clinical score, histologic inflammation, and bone density. Oral LrIbTX and injected IbTX had similar efficacy in treating CIA in rats as measured by clinical joint swelling and histologic inflammation, which were significantly improved versus control bacteria or vehicle injection. No treatments induced measurable levels of anti-IbTX IgG, and there were no differences in macroscopic bone damage or anti-collagen II IgM and IgG levels between CIA groups. Injected IbTX and oral LrIbTX were also equivalent in inhibiting an active delayed-type hypersensitivity (DTH) reaction in rats. Conclusions This work describes the effective oral delivery of a candidate therapeutic peptide, IbTX, via engineered L. reuteri to treat an animal model of autoimmune disease and demonstrates systemic distribution of an intestinally produced peptide. We anticipate that oral delivery of engineered microbes may be a generalizable strategy for enhancing the oral bioavailability of peptide therapeutics.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegβ deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegβ but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg β (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca 2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it’s low levels, HA-Spegβ immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegβ binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegβ display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy. A new mouse model of Spegβ deficiency shows that Spegα prevents the development of dilated cardiomyopathy and decreases atrophy and loss force generation in skeletal muscle. Speg-β interacts with Esd, Fsd2, and Cmya5 and stabilizes interactions among excitation-contraction coupling proteins in triads and dyads.