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Protein That Fights Bacteria and Viruses Cloned by Scientists at The Scripps Research Institute

La Jolla, CA. July 20, 2003 - Bacteria and viruses are completely different classes of pathogens, and not surprisingly the body uses completely different molecular "receptors" to detect them in order to mount an immune defense.

Paradoxically, while the detection systems are different, the actual immune defenses the body employs to clear the system of viral or bacterial infection are much the same. As are the symptoms - to you or me, fighting off bacteria or viruses can produce the same fatigue, inflammation, or hacking cough.

Now a team of researchers at The Scripps Research Institute (TSRI) has published a paper appearing in an upcoming issue of the journal Nature that explains how pathogens as different as viruses and bacteria can have such a common bottom line.

"The proximal reason [for these similar symptoms] is a single protein," says TSRI Professor Bruce Beutler, M.D., who led the research.

This protein, called Trif, associates with different "receptors" that detect a virus or a bacterium on the surfaces of human cells. Trif is a signal transducer - it helps turn these positive detections into immune reactions. Significantly, Trif is the topmost protein shared by the pathway that detects gram-negative bacteria and the pathway that detects most viruses. It is like a waiter who brings orders from two different customers into the same kitchen.

This is the first time that anyone has identified a protein that directly responds to the signals the innate immune system sends when it recognizes both bacteria and viruses.

In addition, Trif could be a potential target for intervening in diseases in which the innate immune system plays a role, such as sepsis. Sepsis basically results from a runaway cascade of inflammation in response to a bacterial infection, and Trif is involved very early in this cascade. If drugs might be designed that could modulate the function of Trif, they might help to improve the prognosis for sepsis.

"You could imagine that blocking this pathway would have a pretty strong anti-inflammatory effect in a diverse range of infectious diseases," says Beutler, who identified and cloned the Trif gene (called Lps2) together with Kasper Hoebe, Ph.D., a postdoctoral fellow in the Beutler laboratory. TSRI Associate Professor Jiahuai Han, Ph.D. and Sung Kim, Ph.D., a postdoctoral fellow in Han's laboratory, collaborated in this effort.

Mapping the Gene

Beutler, Hoebe, and their colleagues mapped the mouse gene Lps2, which has an equivalent gene in humans, after they found a deleterious mutation in a mouse gene that made mouse macrophages unable to sense certain pathogens, thus weakening their innate immune systems.

"Mice that lack this protein are very susceptible to infections like mouse cytomegalovirus," says Beutler, adding that the mice are also unable to respond to bacterial endotoxins, like lipopolysaccharide (LPS) molecules, which are found in the cell walls of many bacteria. In mammals, the innate immune system detects LPS and a multiplicity of other foreign molecules with a family of receptors called the toll-like receptors (TLRs). Mammals have 10 or more different TLR receptors, and one of the goals of scientists like Beutler is to identify how these receptors mediate innate immunity.

Innate immunity is essential for survival in a world filled with microbial pathogens because cells of the innate immune system are the body's first responders, arriving soon after foreign pathogens are detected.

Normally, when human or mouse cells encounter bacteria or viruses, they recognize them with the help of TLRs and other proteins such as Trif. This recognition triggers the immune system, which responds with a multi-stage biochemical defense.

The first stage typically involves the innate immune system and its army of white blood cells, like macrophages, which engulf and destroy pathogens. The macrophages also fight the pathogens by producing chemicals at the site of an infection that induce inflammation. One of these chemicals is called tumor necrosis factor alpha (TNF-alpha). Normally, TNF-alpha is produced in great amounts by macrophages when they are exposed to bacterial and viral "ligands" - the molecules found in the cell walls of bacteria, for instance.

Beutler and his colleagues were able to identify the function of Trif and clone the Lps2 gene after they first observed how a random mutation in one mouse rendered its macrophages unable to produce TNF-alpha when exposed to LPS from gram-negative bacteria like E. coli or when exposed to double-stranded RNA - a product of many viral infections. LPS is known to signal via TLR4: a discovery made by TSRI investigators Beutler and Alexander Poltorak, Ph.D., several years ago. Double-stranded RNA signals via TLR3 - another member of the family. For this reason, Beutler and his coworkers guessed that the mutation might affect a molecule required for both TLR3 and TLR4 to signal properly. They mapped the Lps2 mutation to a 216,000-base pair region of chromosome 17. Of the eight genes in that region, one gene, then called Trif, was a prime candidate because it encoded an adaptor "TIR domain" protein - just the type of protein that might participate in signaling from toll-like receptors.

Beutler and his colleagues sequenced all of the genes in this region and found a mutation affecting a single nucleotide in the TrifLps2 gene. The mutation is a "frameshift" error - the 24 amino acids at the tail end of the gene are exchanged for a completely different set, and when the protein is translated in the cell, it cannot function.

The fact that macrophages with these malfunctioning Trif proteins did not respond to LPS suggests that Trif might make a good target for treating sepsis, which can occur during a widespread bacterial infection. During such an infection, macrophages produce inflammatory chemicals, which help to kill the bacterial cells. But if the systemic endotoxin levels are too high, the macrophages respond by producing a lethal amount of inflammatory chemicals.

Having a way to stop this would be a boon, because the current prognosis for sepsis is dire. It can affect many parts of the body, including the liver, kidneys, heart, intestines, adrenal glands and brain, and death due to septic shock can occur in a matter of hours. According to the Centers for Disease Control and Prevention, sepsis is one of the ten leading causes of both infant and adult mortality in the United States, and, in 1999, directly caused more than 30,000 deaths.

Another interesting conclusion found in the paper is that macrophages with no Trif protein can be divided into two different populations - one pool of cells that are slightly responsive to LPS, and one pool that are unresponsive. This is important because scientists have long considered macrophages to be a homogeneous population of cells.

After observing this, Beutler and his colleagues discovered that even normal macrophages fall into different pools that can be distinguished on the basis of how well they respond to certain stimuli. The apparent heterogeneity might suggest that macrophages specialize somewhat in their function.

"I would guess that some macrophages are better at killing virus-infected cells than at coping with bacteria [and vice-versa]," says Beutler.

The article, "Identification of Lps2 as a key transducer of MyD88-independent TIR signaling" was authored by Kasper Hoebe, Xin Du, Philippe Georgel, Edith Janssen, Koichi Tabeta, Sung Ouk Kim, Jason Goode, Pei Lin, Navjiwan Mann, Suzanne Mudd, Karine Crozat, Sosathya Sovath, Jiahuai Han, and Bruce Beutler and appears in the Advance Online Publication feature of the journal Nature on July 20, 2003. See: The article will appear in print later this year.

The work was funded by grants from the National Institutes of Health, including a multi-center grant from The National Institute of Allergy and Infectious Diseases (NIAID) that has permitted TSRI investigators to study a broad range of problems in innate immunity. Beutler and colleagues hope that this work will be the first of many important discoveries that will result from the approach of creating immune deficiencies by introducing random mutations.

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