| Shigellosis (bacillary dysentery) is one of the most important intestine infection diseases which are epidemic wordwide. Shigella spp. is the causative agent of shigellosis which specifically infect and cause disease in the large intestine of human hosts. Shigella species which are Gram-negative enteric bacteria cause disease by a type III secretion system (T3SS). Type III secretion system (T3SS) is a family of multi-protein complex which serve to secrete macromolecules. Type III secretion (T3SS) systems are used by numerous Gram-negative pathogenic bacteria to deliver effector proteins into cells of their human, animal or plant hosts. The T3SS systems are comprised of the T3SS apparatus, translocators, effectors and specific chaperones.Shigella ?exneri harbours a large virulence plasmid that encodes T3SS responsible for S. ?exneri pathogenesis, through which about 25 effectors are delivered. Some effectors target the actin cytoskeleton to promote entry or inhibit phagocytosis of the bacterium, others promote the spread of the bacterium into adjacent cells or interfere with the host's innate immune responses. Only a small number of these effectors are known to us, most effectors remain to be elucidated. It is reported that IpaH proteins are encoded by plasmid and chromosome of shigella and secreted via the T3SS. Previous studies indicate that five copies of the ipaH genes exist on the large plasmid and seven copies of the ipaH genes exist on the chromosome of S. flexneri 2a 301. All ipaH copies have almost identical C-terminal halves. Although the N-terminal portions of ipaH differ in each gene, they all included a common leucine-rich repeat (LRR) motif. The biological significance of each IpaH protein still largely remains speculative. It has been reported that IpaH7.8 facilitated S. flexneri escape from the vacuole when mouse macrophages or human monocyte-derived macrophages was infected with S. flexneri, IpaH9.8 binded to a splicing factor U2AF35 to modulate host immune response, chromosomal IpaH proteins acted synergistically to modulate the host inflammatory responses. IpaH4.5, another member of IpaH family ecoded by large invasion plasmid, was never reported detailed. Therefore, we systematically explored the role of IpaH4.5 by gene deletion, invasion assay and yeast two-hybrid screening to discover the function of IpaH4.5.To explore the function of ipaH4.5 gene of S. flexneri 2a 301, aλ-Red recombination system was used to knock out this gene first. Then, a pAK plasmid haboring a kanamycin gene was constructed and ipaH4.5gene was subcloned into it to construct a complementary plasmid. A complementary strain was obtained by introducing the complementary plasmid into the deletion mutant. Semi-quantitative RT-PCR results showed that transcription of the ipaH4.5 gene could be detected in the wild-type and complementary strain, but not ipaH4.5 deletion mutant, indicating that ipaH4.5 was successfully deleted in the mutant and restored in the complementary strain.Subsequently, the related phenotypes of wild-type, mutant and complementary strain were compared by some experiments which were carried out as following: 1) the growth curves of wild-type strain, deletion mutant and complementary strain were measured respectively. 2) Some basic biochemical events were also investigated. 3) Survival ability of S. flexneri in extreme conditions such as heat, low pH, high oxygen, and so on. The results of above studies showed no significant differences between three strains, suggesting that the function of this protein might be unrelated to the surviaval ability and metabolism of S. flexneri. Then we performed invasion assays using HeLa cells and murine macrophage cell line J774.A.1, and no difference in invasion ability was observed between the three strains either. After that, quantities of cytokines in the culture supernatants of murine macrophages cell line J774.A.1 after being infected for two hours was measured by enzyme-linked immunosorbent assays (ELISA) for mouse IL-1β, tumor necrosis factor alpha (TNF-а). More IL-1βand TNF-аwere observed in murine macrophages infected with deletion mutant than wild type and complementary strain. So we concluded that IpaH4.5 could inhibit inflammation response of the host. The conjection was further conformed by the murine pneumonia model and the sereny test.In order to explore why the inflammatory response of cell and animals was inhibited by IpaH4.5, we used a dual-luciferase reporter system to detect whether NF-κB signaling was affected by IpaH4.5. NF-κB signaling plays an important role in activating host innate immune responses and is a frequent target of pathogenic effectors. The results show that after transfecting Flag-IpaH4.5 and empty vector into 293T cells, NF-κB signaling was inhibited obviously by IpaH4.5 compared with the empty vector when 293T cells were stimulated by TNF-а. Then intracellular localization of IpaH4.5 was observed by a laser-scanning confocal microscopy, the results showed that ipaH4.5 localized in cytoplasm and nucleus.We also performed yeast two-hybrid screening to indentify IpaH4.5-binding proteins to find out why IpaH4.5 inhibited NF-κB signaling pathway. Full length of IpaH4.5 was used as a bait to screen human spleen cDNA liberary. Finally 52 clones that interacted with IpaH4.5 were identified. Sequence analysis and functional classification showed that these clones were involved in the following functions: apoptosis, immunity regulation, cell adhesion and transcriptional regulation. The candidate protein in which we were most interested was the p65 subunit of NF-κB protein. Full-length cDNA for this protein was then cloned from the cDNA library. The interaction between IpaH4.5 and p65 were confirmed by in vitro GST pull-down assays and in vivo co-immumoprecitipation analysis. Further studies showed that the interacting domain of p65 with IpaH4.5 was located in section between 1~190 of p65. To determine the significance of the interaction between IpaH4.5 and p65, we used dual-luciferase reporter system to verify the interaction. Luciferase activity assays showed IpaH4.5 directly down-regulated the transcriptional activity of NF-κB by binding with p65 subunit.The above results indicate that the function of IpaH4.5 is the same as other reported effectors like OspG, OspF and IpaH9.8 to modulate host innate immune system. Dual-luciferase reporter system and the yeast two-hybrid screening show that IpaH4.5 downregulate the host inflammatory via binding with p65 subunit of NF-κB. Latest researches on the function of the IpaH protein family show that all members of IpaH protein family have E3 ubiquitin ligase activity. But it is still unclear that what kind of host protein do these specific E3 ubiquitin ligase target. Our research has found out about 50 proteins binding with IpaH4.5 by yeast two-hybrid screening. These proteins may be substrates of IpaH-type E3 ubiquitin ligase. Therefore it is necessary to adoption of ubiquitination experiment to prove this conjecture.In conclusion, our data demonstrated that IpaH4.5 directly inhibited NF-κB by interacting with p65 subunit. These findings expand our knowledge of the function of the IpaH family, and provide important clues for understanding the molecular pathogenesis of shihella and new ideas for vaccine development.
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