Structure–function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB

Abstract

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during Salmonella infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic Escherichia coli cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1′ residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1′ residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…EHEC NleC is a metalloprotease capable of reducing the amount of histone acetyltransferase p300 in the nucleus, thereby attenuating the expression of the pro-inflammatory cytokine IL-8 gene [97] ( Figure 2D). Salmonella enterica serovar Typhimurium also secretes metalloproteases: PipA, GogA, and GtgA [98,99]. These enzymes target the nucleus and degrade subunits of NF-κB p65, RelB, and cRel.…”

Salmonella Virulence and Immune Escape

“…Capsules in S. Typhimurium are wrapped around LPS, which also prevents the inflammatory response induced by TLR4 recognition [181]. Even if TLRs successfully identify the PAMPs, Salmonella SPI-2 encoded proteins, i.e., SseL, SpvD, PipA, GogA, GtgA, SpvC, can inhibit the nuclear factor kappa beta (NF-κB), extracellular signal-regulated kinase (Erk), and mitogen-activated protein kinase (MAPK) activation, thus suppressing the transcriptional responses leading to inflammation (Figure 1) [85,106,[184][185][186]. SseL acts as a deubiquitinase and prevents the ubiquitination of IkB-α.…”

Bacterial nucleomodulins: A coevolutionary adaptation to the eukaryotic command center

“…enterica and NleC from E . coli are T3SS-injected nucleomodulins that reportedly contain short metal-binding motifs for active-site zinc [ 66 , 117 ]. While these metalloproteases were identified to localize to the nuclei of transfected and infected cells, there is little known regarding the mechanisms used for nuclear translocation [ 118 , 119 ].…”

“…While these metalloproteases were identified to localize to the nuclei of transfected and infected cells, there is little known regarding the mechanisms used for nuclear translocation [ 118 , 119 ]. Studies have shown that PipA, GtgA, GogA, and NleC redundantly target components involved in the NF-κB signaling pathway [ 117 , 119 , 120 ]. PipA, GogA, and GtgA cleave RelA (p65), RelB, and occasionally cRel nuclear transcription factors, but do not cleave NF-κB1 (p105/p50) or NF-κB2 (p100/p52) [ 117 ].…”

“…Studies have shown that PipA, GtgA, GogA, and NleC redundantly target components involved in the NF-κB signaling pathway [ 117 , 119 , 120 ]. PipA, GogA, and GtgA cleave RelA (p65), RelB, and occasionally cRel nuclear transcription factors, but do not cleave NF-κB1 (p105/p50) or NF-κB2 (p100/p52) [ 117 ]. NleC, on the other hand, reportedly cleaves all 5 NF-κB subunits and is the first bacterial effector described to facilitate cleavage and degradation of histone acetyltransferase p300 [ 3 , 18 , 121 ].…”