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Elucidation of previously unknown offense-defense mechanism in intestinal tract: pathogenic microbe (Shigella) vs. host’s innate immunity (Press Release)

Release Date
12 Mar, 2012
  • BL44XU (Macromolecular Assemblies)
- Steric structure of newly found pathogenic agent, and discovery of the target host factor

Tokyo University

Every year, approximately 15 million people lose their lives due to infectious diseases, and around 2 million out of them are caused by enteral infections. Although the intestinal tract is constantly exposed to numerous microbes, it has layers of protective barriers centered on innate immunity*5 to guard the living system from microbial invasion. On the other hand, the enteropathogenic species Shigella and the allied pathogenic E-coli (typically 157) possess a highly sophisticated mechanism to worm their way into the living body, tactically eluding the protective systems. However, the details of these mechanisms-the innate immunity mechanism blocking pathogenic microbial invasion into intestinal mucosa, and the strategy employed by pathogenic microbes to circumvent them-have been a complete mystery.
Researchers at the Institute of Medical Science of Tokyo University (Prof. Chihiro Sasagawa, Dr.Masato Sanada, et al.) conducted a study, in collaboration with Prof. Tsunehiro Mizushima (University of Hyogo) and the “SPring-8,” on this issue, using Shigella as a model microbe. They elucidated the following mechanisms: (i) the recognition mechanism to detect the invasion of pathogenic microbes into mucosal epithelia, and (ii) the methods Shigella takes to counter the recognition and protection measures taken by the host.

In more specific terms, the results are summarized as below.
(i) A leaf-like protrusion (or, ruffle membrane) is formed when a pathogenic microbe invades a cell. The study demonstrated the importance of the inflammation signal pathway that depends on the diacylglycerol*4 (DAG)-TRAF6-NF-kB pathway localized in the leaf-like protrusion, which provides a mechanism to recognize the leaf-like protrusion as a warning signal.
(ii) The study identified that Shigella secretes OspI as the countermeasure, and clarified the steric structure (Fig. 2) and biochemical properties of the protein.
The researchers discovered, in conducting the study summarized in (i) and (ii), that OspI is a “new deamidating enzyme” that makes a specific binding to UBC13 and deamidates its 100-th glutamine, resulting in the inhibition of TRAF6 activation, and thereby plays an important role in the proper control of the above-described inflammation signal pathway. This study discovered a previously unknown defense mechanism in place in mucosal epithelia*2 that produces an effect against the pathogenic microbes, especially in the early stage of infection, and it also clarified the hidden strategy on the side of pathogenic microbes to evade the mechanism. Such knowledge should provide targets for future research, in view of developing new drugs and vaccines.

"The Shigella effector OspI deamidates Ubc13 to dampen the inflammatory response"
Takahito Sanada1, Minsoo Kim1, Hitomi Mimuro2, Masato Suzuki3, Michinaga Ogawa3, Akiho Oyama3, Hiroshi Ashida3, Taira Kobayashi3, Tomohiro Koyama4, Shinya Nagai4, Yuri Shibata5, Jin Gohda5, Jun-ichiro Inoue5, Tsunehiro Mizushima6*, and Chihiro Sasakawa1, 3, 4*.
1 Department of Infectious Disease Control; International Research Center for Infectious Diseases; University of Tokyo; Minato-ku, Tokyo 108-8639, Japan.
2 Division of Bacteriology; Department of Infectious Disease Control; International Research Center for Infectious Diseases; University of Tokyo; Minato-ku, Tokyo 108-8639, Japan.
3 Division of Bacterial Infection; Department of Microbiology and Immunology; Institute of Medical Science; University of Tokyo; Minato-ku, Tokyo 108-8639, Japan.
4 Nippon Institute for Biological Science; 9-2221-1, Shinmachi, Ome, Tokyo 198-0024, Japan.
5 Division of Cellular and Molecular Biology; Department of Cancer Biology; Institute of Medical Science; University of Tokyo; Minato-ku, Tokyo 108-8639, Japan.
6 Picobiology Institute; Department of Life Science; Graduate School of Life Science; University of Hyogo; 3-2-1 Kouto, Kamigori, Akoh, Hyogo 678-1297, Japan.


Fig.1. Inflammatory suppression mechanism of Shigella through OspI
Fig.1. Inflammatory suppression mechanism of Shigella through OspI

The invasion of Shigella*1 into epithelial cells triggers the formation of a ruffle membrane (leaf-like protrusion; macropinocytosis) in the vicinity of the microbe. The bacteria break the ruffle membrane and finally reach the cellular cytoplasm. As the ruffle membrane formation proceeds in the basolateral surface of epithelial cells, diacylglycerol (DAG) tends to localize in the membrane. Diacylglycerol acts as a second messenger for the signal created by protein kinase C (PKC) and thus has the effect of activating the CMB (CARD-MALT1-BCL10) complex -TRAF6- NF-kB pathway, resulting in induced production of antibacterial peptides, inflammatory cytokine or chemokine.*6 To circumvent this for continued survival and proliferation within the cytoplasm, Shigella secretes an OspI effector (a pathogenic agent) through its type 3 secretion machine*3 when it invades the cell. The OspI makes a specific bonding to UBC13 and converts its 100-th glutamine into a glutamic acid through deamidation, leading to deactivation of the UBC13. As the UBC13 serves as an activating factor of TRAF6, its deactivation hinders the activation of TRAF6, thus disabling the NF-kB signal pathways that lie downstream. Using this scheme, Shigella inhibits the host's inflammation and innate immune reaction immediately after the onset of its infection, resulting in the accelerated proliferation of the pathogenic microbe within the mucosal epithelia.

Fig.2. Steric structure of OspI
Fig.2. Steric structure of OspI

A crystalline sample of OspI protein-secreted through the type 3 secretion machine of Shigella-was successfully prepared and used for crystal structure analysis. The analysis was conducted using SPring-8 (BL44XU), which successfully clarified the steric configuration of OspI in 2.0Å resolution (registered to Protein Data Bank, ID number: 3B21). The analysis proved that OspI had a structure consisting of four β-strands (red), seven α-helixes (blue), and one 310 helix (blue). A comparison of OspI's steric structure with those of other reported proteins in terms of their primary sequences indicated that no known protein had a primary amino acid sequence similar to OspI. However, it revealed that the steric structure of OspI is similar to that of AvrPphB, which is a known etiological agent of Pseudomonas-Syringae (a phytopathogenic fungus with a type 3 secretion machine similar to that of Shigella). The AvrPphB protein is a protease enzyme that has the same type of active center as that of OspI, i.e. a center consisting of cysteine (Csy), histidine (His), and asparagine acid (Asp). By taking advantage of these clues, substitution variants were prepared for each of the amino acids, which then clearly indicated the importance of the three amino-acid residues (i.e. Cys-His-Asp) with respect to Ospl's activity of deamidating UBC13.

*1 Shigella

Shigella (Bacillus dysenteriae) belongs to the same taxonomic class as Escherichia coli, and is the causative microorganism of shigellosis. Last August, a shigella outbreak was triggered in a restaurant chain by an infection of this microorganism. Shigella initiates infection by invading the absorptive epithelial cells for proliferation in the lower intestine (large intestine and rectum), and spreads into the neighboring epidermal cells. The infection is detected by innate immunity, causing inflammation inside the intestinal tract.

*2 Mucosal epithelia
Monolayer epithelial cells on the surface of luminal organs (e.g. intestinal tract) play an important role in the defense mechanism (a barrier against microbial invasions), as well as in the absorption of nourishment from intraluminal surfaces.

*3 Type 3 secretion machine
The pathogenic agent (effector) secretion complex innately present in many causative bacteria.

*4 Diacylglycerol
A molecule consisting of two fatty acid molecules covalently bonded to a glycerol molecule through ester linkage. It is localized in a host cell membrane and acts as a second messenger.

*5 innate immunity
The innate immunity system that naturally exists in our body. It constitutes a defense system peculiar to a living body that recognizes and precludes the invasion of disease-causing agents such as microorganisms.

*6 Inflammatory cytokine, chemokine
Any of the low molecular weight proteins involved in detecting the invasion of disease causing agents (or an incidence of heterogenized body of its own) and sending signals to surrounding cells and tissues, thus triggering an activation of the (inflammatory) immune system. These proteins have the effect of gathering neutrophils and hematopietic cells to the inflammation site.

For more information‚ please contact:
 Prof. Chihiro Sasagawa (The Institute of Medical Science of Tokyo University)

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