Shigella flexneriT3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-κB p65 protein

Abstract

SummaryShigella species possess a type III secretion system (T3SS), which is required for human infection and that delivers effector proteins into target host cells. Here, we show that the effector, IpaH4.5 dampens the pro-inflammatory cytokine response. In both the Sereny test and a murine lung infection model, the Shigella DipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro-inflammatory cytokine levels (MIP-2 and TNF-a) in the lung homogenates of wild type-infected mice. Moreover, there was a threefold decrease in bacterial colonization of the mutant compared with the WT and DipaH4.5/ ipaH4.5-rescued strains. Yeast two-hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF-kB. Ten truncated versions of IpaH4.5 and p65 spanning different regions were constructed and expressed to further map the IpaH binding sites with p65. The results revealed that the p65 region spanning amino acids 1-190 of p65 interacted with the IpaH4.5/1-293 N-terminal region. In vitro, IpaH4.5 displayed ubiquitin ligase activity towards ubiquitin and p65. Furthermore, the transcriptional activity of NF-kB was shown to be inhibited by IpaH4.5 utilizing a dual-luciferase reporter gene detection system containing NF-kB promoter response elements. Thus, we conclude that the IpaH4.5 protein is an E3 ubiquitin ligase capable of directly regulating the host inflammatory response by inhibiting the NF-kB signalling pathway.

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

“…This is especially important because the apparent redundancy of at least the chromosomal genes suggests that only quantitative ablation of IpaH activity will result in observable changes in the host immune response (16). Consistent with this conclusion, two identified targets for IpaH ligases, NEMO for IpaH9.8 and NF-B p65 for IpaH4.5, participate in the same host innate immune response pathway (23,31). With variable ubiquitination targets, inhibiting IpaH target interactions would be inadequate to inhibit the downstream consequences of IpaH ligase activity.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…102 Certain NEL family members, including IpaH9.8, IpaH0722 and IpaH4.5, have been shown to tamper with the host NF-κB cascade by ubiquitinating NEMO, TRAF2 and p65 for proteasomal degradation, respectively. [103][104][105] Another NEL effector, IpaH7.8, possesses E3 ligase activity in vitro and targets an inhibitor of Cullin-based RING E3 ligase named GLMN for degradation, which augments inflammasome activation. 106 In addition, OspG and OspI, belonging to a different subsets of S. flexneri effectors, have recently been identified to suppress NF-κB activation by disrupting the ubiquitin system.…”

Bacterial E3 Ubiquitin Ligase IpaH4.5 of Shigella flexneri Targets TBK1 To Dampen the Host Antibacterial Response

“…6C). A previous study revealed that IpaH4.5 interacted with p65 to inhibit the host-inflammatory response and promote bacterial colonization of host cells (23). To further dissect the roles of the TBK1/ IRF3 pathway and the impact of TBK1 degradation induced by IpaH4.5 on the inflammatory response and bacterial proliferation, we first sought to determine whether the IRF3 pathway could regulate the proliferation of Shigella bacteria.…”

“…The effectors of the IpaH family produced by mammal, fish, and plant pathogenic bacteria share several structural and functional characteristics (18) and have a highly conserved N-terminal leucine-rich repeat (LRR) domain and a C-terminal region with E3 ubiquitin (Ub) ligase activity found in pathogenic and symbiotic bacteria (19)(20)(21)(22). Recently, IpaH4.5 was found to target NF-kB subunit p65 to downregulate the host immune response (23). In this study, we identified TBK1 as a novel host target protein of IpaH4.5.…”