Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence

Abstract

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms.

Regulation, Function, and Detection of Protein Acetylation in Bacteria

“…Lrp is regulated by Pat and CobB in vitro (101), and both a K36Q variant and a fully acetylated protein (method discussed later) led to severe reductions in DNA-binding activity. PhoP is acetylated by Pat at K201 and is deacetylated by CobB (102). Excitingly, a K201Q mutation led to attenuated growth in macrophages and virulence in mouse models.…”

“…A number of virulence factors are acetylated, but the significance of these findings needs to be explored. So far, acetylation likely affects the virulence of Erwinia amylovora, Pseudomonas aeruginosa, Salmonella spp., and M. tuberculosis (32,38,44,46,102,103). With an alarming rise in antibiotic drug resistance, there is an imperative need for new drug targets.…”

The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium

“…Expression and purification of RhaR, STM14_1074, NrdF and FliT were performed as Lrp. The protocols to purify Pat and CobB were described previously (Ren et al, 2016). …”

“…The Pat-mediated in vitro acetylation reaction was performed as described previously (Ren et al, 2016). The in vitro acetylation reaction buffer containing 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol and 10 mM sodium butyrate.…”

“…The CobB-mediated in vitro deacetylation reaction was performed as described previously (Ren et al, 2016). Deacetylation reaction by CobB was performed in deacetylation reaction buffer including 50 mM Tris-HCl (pH 8.0), 135 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 in the presence or absence of 1 mM NAD + , and in the presence or absence of 10 mM nicotinamide (NAM), an inhibitor for CobB (Bitterman et al, 2002).…”