Shigella flexneri T3SS effector IpaH4.5 modulates the host inflammatory response via interaction with NF-κB p65 protein

Cell Microbiol. 2013 Mar;15(3):474-85. doi: 10.1111/cmi.12052. Epub 2012 Nov 13.


Shigella 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 ΔipaH4.5 mutant strain caused more severe inflammatory responses and significantly induced higher pro-inflammatory cytokine levels (MIP-2 and TNF-α) 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 ΔipaH4.5/ipaH4.5-rescued strains. Yeast two-hybrid screening showed that IpaH4.5 specifically interacts with the p65 subunit of NF-κB. 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 thatthe 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-κB was shown to be inhibited by IpaH4.5 utilizing a dual-luciferase reporter gene detection system containing NF-κB 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-κB signalling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Bacterial / genetics
  • Antigens, Bacterial / immunology
  • Antigens, Bacterial / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism*
  • Cell Line
  • DNA Mutational Analysis
  • Disease Models, Animal
  • Dysentery, Bacillary / microbiology
  • Dysentery, Bacillary / pathology
  • Guinea Pigs
  • Host-Pathogen Interactions*
  • Humans
  • Immune Evasion*
  • Keratitis / microbiology
  • Keratitis / pathology
  • Mice
  • Pneumonia, Bacterial / microbiology
  • Pneumonia, Bacterial / pathology
  • Protein Binding
  • Protein Interaction Mapping
  • Shigella flexneri / genetics
  • Shigella flexneri / immunology*
  • Shigella flexneri / pathogenicity*
  • Transcription Factor RelA / antagonists & inhibitors*
  • Transcription Factor RelA / immunology
  • Transcription Factor RelA / metabolism
  • Two-Hybrid System Techniques
  • Virulence Factors / genetics
  • Virulence Factors / immunology
  • Virulence Factors / metabolism*


  • Antigens, Bacterial
  • Bacterial Proteins
  • Transcription Factor RelA
  • Virulence Factors
  • ipaH protein, Shigella flexneri