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NF-κB is crucial for innate immune defence against microbial infection (1,2). Inhibition of NF-κB signalling has been observed with various bacterial infections (3,4). The NF-κB pathway critically requires multiple ubiquitin-chain signals of different natures (5,6). The question of whether ubiquitin-chain signalling and its specificity in NF-κB activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-κB signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-κB signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-κB activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling., Bacterial pathogens such as Salmonella, Shigella and enteropathogenic E. coli (EPEC) target the NF-κB pathway to counteract host defence (3,4,7). Recent genetic studies reveal that EPEC suppression of host NF-κB [...]

NF-[kappa]B is critical in innate immune defense responses against invading microbial pathogens. Legionella pneumophila infection of lung macrophages causes Legionnaire's disease with pneumonia symptoms. A set of NF-[kappa]B-controlled genes involved in inflammation and anti-apoptosis are up-regulated in macrophages upon L. pneumophila infection in a Legionella Dot/Icm type IV secretion system-dependent manner. Among [approximately equal to]100 Dot/Icm substrates screened, we identified LegK1 as the sole Legionella protein that harbors a highly potent NF-[kappa]B-stimulating activity. LegK1 does not affect MAPK and IFN pathways. Activation of the NF-[kappa]B pathway by LegK1 requires its eukaryotic-like Ser/Thr kinase activity and is independent of upstream components in the NF-[kappa]B pathway, including TRAFs, NIK, MEKK3, and TAK1. Cell-free reconstitution revealed that LegK1 stimulated NF-[kappa]B activation in the absence of IKK[alpha] and IKK[beta], and LegK1 efficiently phosphorylated I[kappa]B[alpha] on Ser-32 and Ser-36 both in vitro and in cells. LegK1 seems to mimic the host IKK as LegK1 also directly phosphorylated other I[kappa]B family of inhibitors including p100 in the noncanonical NF-[kappa]B pathway. Phosphorylation of p100 by LegK1 led to its maturation into p52. Thus, LegK1 is a bacterial effector that directly activates the host NF-[kappa]B signaling and likely plays important roles in modulating macrophage defense or inflammatory responses during L. pneumophila infection. innate immunity | Legionella pneumophila | p100 processing | Type IV secretion | bacterial pathogenesis

NF-?B is crucial for innate immune defence against microbial infection. Inhibition of NF-?B signalling has been observed with various bacterial infections. The NF-?B pathway critically requires multiple ubiquitin-chain signals of different natures. The question of whether ubiquitin-chain signalling and its specificity in NF-?B activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-?B signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-?B signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-?B activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-?B signalling. [ABSTRACT FROM AUTHOR]