Britta Körner

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 metalloproteinase 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.

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.

The type VI secretion system (T6SS) is a complex secretion system encoded by many Gram-negative bacteria to translocate effector proteins directly into target cells. Due to its high complexity and energy-intensive firing process, regulation of the T6SS is tightly controlled in many organisms. Y. pseudotuberculosis encodes four complete T6SS clusters but lacks genes implicated in T6SS gene regulation in other microorganisms, indicating a distinct control mechanism. Here, we could show that the T6SS4 of Y. pseudotuberculosis is heterogeneously expressed within a population, which is determined by the transcriptional T6SS4 activator RovC. Moreover, the T6SS4 and RovC are embedded in a complex and global regulatory network, including the global post-transcriptional regulator CsrA, the Yersinia modulator A (YmoA), the global protease Lon, and RNases (PNP and RNase III). Post-transcriptional processing of the T6SS4 polycistron and different transcript stability within the operon also achieve a higher regulatory complexity. In summary, our work provides new insights into the sophisticated and complex regulatory network of the T6SS4 of Y. pseudotuberculosis, which clearly differs from regulation in other organisms.