Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates prior to secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.

A factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) is a representative of the ␥-butyrolactone autoregulators that trigger secondary metabolism and morphogenesis in the Gram-positive, filamentous bacterial genus Streptomyces. Here, we report the A factor biosynthesis pathway in Streptomyces griseus. The monomeric AfsA, containing a tandem repeat domain of Ϸ80 aa, catalyzed ␤-ketoacyl transfer from 8-methyl-3-oxononanoyl-acyl carrier protein to the hydroxyl group of dihydroxyacetone phosphate (DHAP), thus producing an 8-methyl-3-oxononanoyl-DHAP ester. The fatty acid ester was nonenzymatically converted to a butenolide phosphate by intramolecular aldol condensation. The butenolide phosphate was then reduced by BprA that was encoded just downstream of afsA. The phosphate group on the resultant butanolide was finally removed by a phosphatase, resulting in formation of A factor. The 8-methyl-3-oxononanoyl-DHAP ester produced by the action of AfsA was also converted to A factor in an alternative way; the phosphate group on the ester was first removed by a phosphatase and the dephosphorylated ester was converted nonenzymatically to a butenolide, which was then reduced by a reductase different from BprA, resulting in A factor. Because introduction of afsA alone into Escherichia coli caused the host to produce a substance having A factor activity, the reductase(s) and phosphatase(s) were not specific to the A factor biosynthesis but commonly present in bacteria. AfsA is thus the key enzyme for the biosynthesis of ␥-butyrolactones.A factor ͉ Streptomyces griseus ͉ AfsA ͉ cell-to-cell signaling

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In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-␥-butyrolactone) switches on aerial mycelium formation and secondary metabolite biosynthesis. An A-factor-dependent transcriptional activator, AdpA, activates multiple genes required for morphological development and secondary metabolism in a programmed manner. A region upstream of a zinc-containing metalloendopeptidase gene (sgmA) was found among the DNA fragments that had been isolated as AdpA-binding sites. The primary product of sgmA consisted of N-terminal pre, N-terminal pro, mature, and C-terminal pro regions. sgmA was transcribed in an AdpA-dependent manner, and its transcription was markedly enhanced at the timing of aerial mycelium formation. AdpA bound two sites in the region upstream of the sgmA promoter; one was at about nucleotide position ؊60 (A site) with respect to the transcriptional start point of sgmA, and the other was at about position ؊260 (B site), as determined by DNase I footprinting. Transcriptional analysis with mutated promoters showed that the A site was essential for the switching on of sgmA transcription and that the B site was necessary for the marked enhancement of transcription at the timing of aerial mycelium formation. Disruption of the chromosomal sgmA gene resulted in a delay in aerial hypha formation by half a day. SgmA is therefore suggested to be associated with the programmed morphological development of Streptomyces, in which this peptidase, perhaps together with other hydrolytic enzymes, plays a role in the degradation of proteins in substrate hyphae for reuse in aerial hypha formation.Members of the gram-positive, soil-inhabiting, filamentous bacterial genus Streptomyces produce a wide variety of secondary metabolites and show complex morphological differentiation culminating in sporulation. These characteristics make members of this genus important as industrial microorganisms and as one of the model prokaryotes for studying multicellular differentiation (3, 4). On agar medium, one or more germ tubes formed from a germinating spore grow into substrate hyphae, which branch frequently and grow rapidly by cell wall extension at the hyphal tips. Subsequently, aerial hyphae emerge by reuse of material assimilated into the substrate mycelium, such as DNA, proteins, and storage compounds. Many cells in substrate hyphae thus lyse and die (5,21,31). At this stage of development, therefore, enzymes for the degradation of these substances, such as proteases, nucleases, lipases, and glucanases, are presumably required. In fact, nucleases and serine proteases are associated with the development of aerial hyphae from substrate hyphae (5, 23). When the apical growth of aerial hyphae stops, in contrast to the substrate mycelium, septa are formed at regular intervals along the hyphae to form many unigenomic compartments within a sheath. The sporulation septa consist of two membrane layers separated by a double layer of cell wall material, which permits the eventual separation of adjacent spores (7). Spore chains usua...

Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput assay for type III protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs can attenuate this key virulence pathway without affecting bacterial growth. Amongst the active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis, targets S. Typhimurium pathogenicity island-1 (SPI-1) type III secretion system (T3SS) effectors and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids present in other TCMs, such as quercetin also inactivated the SPI-1 T3SS and attenuated S. Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can directly interfere with key bacterial virulence pathways and reveals a previously unappreciated mechanism of action for anti-infective medicinal plants.