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Targeting the histone code as a bacterial strategy for selectively modulating gene expression

a bacterial strategy and gene expression technology, applied in the field of targeting the histone code as a bacterial strategy for selectively modulating gene expression, can solve the problems of excessive inflammation, ineffective invading the apical pole of polarized iec, and detrimental to i>shigella/i>'s survival in the hos

Inactive Publication Date: 2007-04-05
INST PASTEUR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008] The Examples provided herein show that the ospF gene encodes a phosphatase and that this phosphatase can interfere with signalling pathways involving extracellular signal regulated kinases (Erks) and mitogen-activated protein kinases (MAPKs). The Examples below also show that OspF phosphatase can decrease the transcription of a variety of host genes, including those encoding c-fos and IL-8. For IL-8 expression, the Examples demonstrate that the OspF phosphatase decreases transcription of the IL-8 gene by preventing changes in chromatin structure needed to allow transcription proteins like NF-κB and RNA polymerase II (RNAPII) to access the IL-8 promoter. Furthermore, the Examples also show that OspF phosphatase can downregulate the expression of interleukin 12 (IL-12), leading to a Th2 type adaptive immune response.

Problems solved by technology

In contrast to Salmonella typhimurium, Shigella is inefficient at invading the apical pole of polarized IEC.
Thus, triggering excessive inflammation is detrimental to Shigella's survival in the host.

Method used

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  • Targeting the histone code as a bacterial strategy for selectively modulating gene expression
  • Targeting the histone code as a bacterial strategy for selectively modulating gene expression
  • Targeting the histone code as a bacterial strategy for selectively modulating gene expression

Examples

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example 1

Materials and Methods

[0086] Cell Culture. The human intestinal epithelial cell line Caco-2, derived from a colonic carcinoma, and HeLa cells were used. Synchronization of the cells by a double thymidine block was performed as described previously (Harper, 2005).

[0087] Bacterial strains. The wild-type (WT) invasive strain of S. flexneri serotype 5a and a noninvasive variant (VP−) of the WT strain lacking the 220 kB virulence plasmid were used. The ipa mutants strains have been described previously (Mavris et al., 2002a). To construct the ospF mutant, a PCR amplified DNA fragment encompassing nucleotides 61 to 420 of ospF was cloned between the XbaI and EcoRI sites of the suicide plasmid pSW23T, generating pSWOspFTr. This plasmid was transferred by conjugation to the WT strain WT-Sm. A plasmid expressing the ospF gene was constructed by inserting a PCR fragment encompassing the ospF gene between the EcoRI and HindIII sites of pUC18, forming pUC18-OspF. This plasmid was used to compl...

example 2

Shigella Inactivates Erk and Sequesters Erk into the Nucleus

[0098] MAPK pathways play important roles in regulating gene expression in eukaryotic cells by phosphorylating transcription factors, co-regulators, and chromatin remodelling components. The three major subfamilies of MAPKs in mammalian cells include the Erk 1 and 2, JNK 1, 2, and 3, and p38 proteins (p38α / β / γ / δ). MAPK kinases (MAPKKs) activate MAPKs by phosphorylating threonine (Thr) and tyrosine (Tyr) residues at TEY sequences located in the MAPK's activation loop domain. MAPKKs are in turn activated by phosphorylation at serine (Ser) and Thr residues performed by MAPKK kinases (MAPKKKs). In quiescent cells, the two isoforms of MAPKs, Erk1 and Erk2, are inactive and retained in the cytoplasm via a direct association with their upstream activator, the kinase MEK1. Upon cell activation, activated MEK1 phosphorylates Erk1 and Erk2 on the TEY sequence leading to dissociation of the MEK-Erk complex before Erk translocates int...

example 3

The ospF Gene Encodes a Bacterial Phosphatase that Directly and Dually Dephosphorylates Erk

[0103] Upon contact with the epithelial cell surface, the Shigella TTSS apparatus activates and approximately 20 proteins are secreted through this secreton. The TTSS apparatus is weakly active during bacterial growth in vitro at 37° C. and is deregulated by inactivation of the ipaB or ipaD genes (Menard et al., 1994).

[0104] Since the major Erk phosphatases described in mammals are soluble enzymes, a strategy was developed to identify a putative bacterial DSP using bacterial supernatants from deregulated mutant strains in which the ipaB gene has been either inactivated (ipaB2 strain) or disrupted, leading to a truncated version of the ipaB protein (ipaB4 strain) (Mavris et al., 2002a). Supernatants from these mutant strains were assayed for the phosphatase activity using a commercial phospho-Erk2-glutathione S transferase (pErk2-GST) fusion peptide as a substrate.

[0105] The supernatant from...

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Abstract

The ospF gene of Shigella flexneri encodes a phosphatase, which is a member of a new class of phosphatases. The OspF phosphatase inhibits the activity of several proteins either by direct protein modification or transcription downregulation. These proteins include MAP kinase, IL-8, CCL20, IL-12, AP1, CREB, RPA p32, and BCL2 related proteins. Methods for treating diseases using OspF phosphatase, methods for identifying agents that modulate OspF phosphatase's activity, methods for identifying agents that mimic OspF phosphatase's activity, and immunogenic compositions comprising OspF phosphatase are provided. A strain of Shigella flexneri containing an inactivated ospF gene is also provided.

Description

[0001] This application claims the benefit of U.S. provisional application No. 60 / 716,931, filed on Sep. 15, 2005, which is incorporated herein by reference.FIELD OF THE INVENTION [0002] This invention relates to a member of a new class of dual specific phosphatases, methods for treating diseases using the phosphatase, methods for identifying agents that modulate the phosphatase's activity, methods for identifying agents that mimic the phosphatase's activity, a method for regulating an immune response, and immunogenic compositions comprising the phosphatase. The invention also relates to a strain of Shigella flexneri containing an inactivated ospF gene and a vaccine comprising a strain of Shigella flexneri containing an inactivated ospF gene. BACKGROUND OF THE INVENTION [0003]Shigella flexneri is a Gram-negative bacterial pathogen that causes bacillary dysentery in humans by infecting epithelial cells of the colon. Shigella primarily infects intestinal epithelial cells (IECs) (Jung ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/00G01N33/574
CPCA61K38/164A61K38/45A61K39/0283A61K2039/521C07K14/4702C07K14/4703C07K14/4747C07K14/5421C07K14/5434C12N9/1205C12N9/16A61P29/00A61P31/04A61P35/00A61P37/06Y02A50/30
Inventor SANSONETTI, PHILIPPEARBIBE, LAURENCEPARSOT, CLAUDEKIM, DONG WOOKPHALIPON, ARMELLE
Owner INST PASTEUR
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