Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which—in contrast to the direct cytoplasmic injection of T3SS effector proteins—are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida (Phdp). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1β. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

Summary Pseudomonas putida can be used as a host for the autotransporter‐mediated surface display of enzymes (autodisplay), resulting in whole‐cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter‐mediated secretion depends on the N‐terminal signal peptide as well as on the C‐terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole‐cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12‐fold enhanced to 638 mU ml−1 OD −1 compared with the signal peptide of V. cholerae CtxB (52 mU ml−1 OD −1). This positive effect was confirmed with a β‐glucosidase as a second example enzyme. Here, cells expressing the protein with N‐terminal OprF signal peptide showed more than fourfold higher β‐glucosidase activity (181 mU ml−1 OD −1) than with the CtxB signal peptide (42 mU ml−1 OD −1). SDS‐PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.