A substrate-fusion protein is trapped inside the Type III Secretion System channel in Shigella flexneri.

The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

Potent Rifampicin derivatives can clear MRSA infections at single low doses when concomitantly dosed with Vancomycin.

For a number of years, antimicrobial resistance (AMR) has been a critical issue for humanity. Drug discovery efforts have been very limited and the spread of bacterial pathogens has over-run our traditional arsenal of antibiotics. Bacteria can involve to evade compounds that can halt their rapid growth. The authors have discovered a potent macrocycle derivative that when dosed concomitantly with the standard of care (SOC) antibiotic vancomycin, can clear methicillin resistant Staphylococcus aureus (MRSA) infections. In addition, we have probed the lead compounds in Salmonella typhimurium bacterial strains. In vitro, in vivo, and ADME data have been included to stress the virtues of this new antibiotic.