Arginine GlcNAcylation and Activity Regulation of PhoP by a Type III Secretion System Effector in Salmonella

Abstract

Salmonella type III secretion system (T3SS) effector SseK3 is a glycosyltransferase delivered directly into the host cells to modify host protein substrates, thus manipulating host cellular signal transduction. Here, we identify and characterize the Arg-GlcNAcylation activity of SseK3 inside bacterial cells. Combining Arg-GlcNAc protein immunoprecipitation and mass spectrometry, we found that 60 bacterial proteins were GlcNAcylated during Salmonella infection, especially the two-component signal transduction system regulatory protein PhoP. Moreover, the Arg-GlcNAcylation of PhoP by SseK3 was detected in vivo and in vitro, and four arginine residues, Arg65, Arg66, Arg118, and Arg215 were identified as the GlcNAcylation sites. Site-directed mutagenesis showed that the PhoP R215A change significantly reduced the DNA-binding ability and arginine to alanine change at all four sites (PhoP 4RA) completely eliminated the DNA-binding ability, suggesting that Arg215 is essential for the DNA-binding activity of PhoP and GlcNAcylation of PhoP affects this activity. Additionally, GlcNAcylation of PhoP negatively regulated the activity of PhoP and decreased the expression of its downstream genes. Overall, our work provides an example of the intra-bacterial activities of the T3SS effectors and increases our understanding of endogenous Arg-GlcNAcylation.

Arginine glycosylation regulates UDP-GlcNAc biosynthesis in Salmonella enterica

“…The work described here provides the first evidence that Arg-glycosylation of the bacterial transcription factor NagC by SseK1 increases NagC affinity for DNA. These data also represent, to our knowledge, along with the recently described work regarding Arg-glycosylation of PhoP by SseK3 19 , the first example of a T3SS effector directly regulating gene expression by modifying a transcription factor. Future experiments aiming to solve the co-crystal structure of an Arg-GlcNac-NagC-DNA complex may permit a molecular understanding of how Arg-glycosylation of transcription factors alters their interactions with DNA.…”

“…To some extent, these results are counter-intuitive, because one might reasonably expect that glycosylating a basic amino acid would tend to reduce protein affinity for DNA. We also note that, in a related system, the recent analysis of SseK3 glycosylation of PhoP concluded that there was a slight reduction in PhoP affinity for DNA as a function of PhoP R215 glycosylation, although the binding affinities were not calculated 19 . However, it is not clear from these studies whether the investigators used phosphorylated PhoP or recombinant, non-phosphorylated PhoP for their EMSAs, a variable which might affect the interpretation of these data.…”

“…SseK1 Arg-glycosylates and enhances the activity of several enzymes (GloA, GloB, GloC, and YajL) involved in methylglyoxal detoxification 18 . It was recently described that SseK3-mediated Arg-glycosylation also plays an important role in modulating the DNA-binding activity of Salmonella PhoP, suggesting a mechanism for how Arg-glycosylation may also act as a regulator of gene transcription 19 .…”

Proteomic Analysis Revealed Metabolic Inhibition and Elongation Factor Tu Deamidation by p-Coumaric Acid in Cronobacter sakazakii

“…The protein samples for LC-MS/MS analysis were prepared according to the protocols described previously with minor modifications ( Xue et al, 2022 ). Specifically, the protein sample (50 μg, qualified by optical density at 260 nm) was denatured by adding one-fourth 8M urea (Aladdin, China).…”

“…The digested peptide samples were analyzed using a Q Exactive Plus mass spectrometer and an EASY nano Liquid chromatography (EASY nLC 1200, Thermo Scientific) with an EASY nanoelectrospray interface according to the methods described before ( Xue et al, 2022 ). The nano liquid chromatography system was equipped with a Thermo Scientific Acclaim Pepmap nano-trap column (C18, 5 μm, 100 Å, 100 μm × 2 cm) and a Thermo Scientific EASY-Spray column (Pepmap RSLC, C18, 2 μm, 100 Å, 50 μm × 15 cm).…”