Salmonella effector SteE converts the mammalian serine/threonine kinase GSK3 into a tyrosine kinase to direct macrophage polarization.

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment

Seed Regeneration Potential of Canopy Gaps at Early Formation Stage in Temperate Secondary Forests, Northeast China

Promoting the seed regeneration potential of secondary forests undergoing gap disturbances is an important approach for achieving forest restoration and sustainable management. Seedling recruitment from seed banks strongly determines the seed regeneration potential, but the process is poorly understood in the gaps of secondary forests. The objectives of the present study were to evaluate the effects of gap size, seed availability, and environmental conditions on the seed regeneration potential in temperate secondary forests. It was found that gap formation could favor the invasion of more varieties of species in seed banks, but it also could speed up the turnover rate of seed banks leading to lower seed densities. Seeds of the dominant species, Fraxinus rhynchophylla, were transient in soil and there was a minor and discontinuous contribution of the seed bank to its seedling emergence. For Quercus mongolica, emerging seedling number was positively correlated with seed density in gaps (R = 0.32, P<0.01), especially in medium and small gaps (<500 m2). Furthermore, under canopies, there was a positive correlation between seedling number and seed density of Acer mono (R = 0.43, P<0.01). Gap formation could promote seedling emergence of two gap-dependent species (i.e., Q. mongolica and A. mono), but the contribution of seed banks to seedlings was below 10% after gap creation. Soil moisture and temperature were the restrictive factors controlling the seedling emergence from seeds in gaps and under canopies, respectively. Thus, the regeneration potential from seed banks is limited after gap formation

Structural basis for the glycosyltransferase activity of the Salmonella effector SseK3

The Salmonella secreted effector SseK3 translocates into host cells, targeting innate immune responses including NF-κB activation. SseK3 is a glycosyltransferase that transfers an N-acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolysed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DXD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine-GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural re-arrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free N-acetyl α-D-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine N-glycosylation reaction. Homology models of SseK1, SseK2, and the Escherichia coli orthologue NleB1, reveal differences in the surface electrostatic charge distribution possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine N-glycosyltransferase provides an important step towards a better understanding of this enzyme class and their roles as bacterial effectors

The <i>Salmonella</i> Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3 and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate STAT3 on tyrosine-705. This results in STAT3 activation and both GSK3 and STAT3 are required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker IL-4Rα. Overall, the conversion of GSK3 to a tyrosine-directed kinase represents an unprecedented example of how a bacterial virulence protein reprograms innate immune signalling to establish an anti-inflammatory environment