$11.4 Million CDC Grant Goes to TSRI to Study Immune Response to Anthrax Toxins

The U.S. Centers for Disease Control and Prevention (CDC) has awarded a group of researchers at The Scripps Research Institute (TSRI) a multi-year, $11.4 million grant to study the interaction of the human immune system with toxins of the bacterium Bacillus anthracis, the causative agent of the disease anthrax.

The goal of the grant is to understand how B. anthracis toxins suppress immune responses in humans, circumventing the usual mechanisms by which the body would destroy the bacterium.

Normally, when humans are infected with a bacterium, innate immune cells and other components of the immune system mount a vigorous defense and clear the body of infection. However, B. anthracis is able to defend itself from being killed through the protection of its highly inert outer capsule and through the production of special toxins, which shut off parts of the immune system.

The importance of these toxins is well established, since strains of the bacterium that do not express these toxins are much less virulent. While the identity and structure of the toxins is known by now, how they modulate cell function and allow the bacteria to evade immune surveillance is not really understood at the molecular and biochemical levels.

"It's clear that when someone is infected with Bacillus anthracis, [the bacterium] is able to suppress the immune system of that person," says TSRI Professor Gary Bokoch, who is one of the lead investigators on the grant. But how?

Bokoch and several other TSRI scientists will define genetic and cellular elements that determine susceptibility to anthrax infection and progression, which should facilitate the translation of basic research into clinical applications.

The program project grant is one of several recently announced by CDC Director Dr. Julie Gerberding to fund research grants in biodefense and emerging infectious diseases. These awards are part of the United States Department of Health & Human Service's efforts to build and sustain a robust and long-term program for biodefense research. The duration of funding for the grant is five years, subject to congressional appropriations.

Anthrax and the Immune System

Anthrax is a deadly disease that is caused by infection with the bacterium Bacillus anthracis. It is an ancient disease—both Homer and Virgil wrote about a disease that was probably anthrax.

The Greeks named the disease anthrax, which means coal, because of the characteristic black ulcers that form on the skin of people and animals infected with the bacterium. This cutaneous form of the disease was responsible for widespread outbreaks among livestock through the centuries, and Louis Pasteur famously demonstrated the first anthrax vaccine in 1881, which helped confirm the germ theory of disease.

However, despite the centuries of human and B. anthracis coexistence, little is known about the pathogenic basis of anthrax.

"It's amazing to me that for as long as this disease has been around, there's not a lot understood about the basic mechanisms of how it affects the cells of the immune system," says Bokoch.

Understanding how B. anthracis causes disease is a major public health priority today, particularly since aerosolized anthrax spores cause inhalation anthrax, the most deadly form of the disease, and have the potential to be used as a bioterror weapon.

The TSRI team is not studying these deadly aerosolized spores directly, but is focusing on the interaction of B. anthracis toxins with the immune system that occurs during inhalation anthrax.

In inhalation anthrax, B. anthracis is inhaled as a spore. B. anthracis naturally forms spores when conditions are not right for the bacterium to replicate. When it converts into a spore, it can lie dormant inside its protective, almost indestructible protein coat. When spores of anthrax are breathed in, they are taken up through the lungs by cells called macrophages. The macrophages transport ingested spores to other parts of the body, where they germinate into bacteria and begin reproducing and making toxins.

Two toxins in particular are known to interact with the host immune system. These are the "lethal toxin," which is a metalloprotease (an enzyme that chops up other proteins), and the "edema toxin", which is an adenylate cyclase (a protein that makes cAMP, an important "second messenger" molecule in the body that has a variety of systemic effects).

The lethal toxin, in particular, seems to have the ability to inhibit the immune system. "It shuts off these [immune] responses that would normally be used by the body to kill the bacteria," says Bokoch.

It is not known how the toxins do this. Most of their targets in human cells and their mechanisms of action have not been identified.

The Program Project Grant

The program project grant is designed to be an interactive collaboration among various TSRI investigators. The overall goal of the grant is to elucidate the molecular targets and mechanisms by which B. anthracis suppresses the innate immune responses in humans and to determine susceptibility to anthrax infection and progression.

The grant seeks to define these mechanisms at several different levels—genetic, biochemical, and physiological—and is composed of several different projects that approach the question from different angles. A core laboratory, directed by TSRI Associate Professors Ulla Knaus and Marta Perego, will be established to produce necessary reagents.

One thing that is not clearly understood is why some people are more susceptible to infection than others, and some of the projects in the grant will address this question. TSRI Professor Bruce Beutler, for instance, will investigate which host genes are required for susceptibility and/or resistance to the anthrax lethal toxin.

"The anthrax lethal toxin has been identified," says Beutler. "But the mechanism of toxicity is very poorly understood. We hope to find proteins in the pathway that leads to killing of macrophages."

Similarly, another project on the grant will look for polymorphisms—DNA variations that differ from person to person—that might make some people more susceptible to anthrax than others.

TSRI Associate Professor Bruce Zuraw, will look at the interaction of B. anthracis toxins with human alveolar macrophages—a type of innate immune cell that normally clears the lungs of bacteria and other particles by engulfing and destroying them. During inhalation anthrax, most B. anthracis spores are destroyed by these macrophages, but a few may survive and use the macrophages as a vehicle to spread within the body. The macrophages shuttle the ingested B. anthracis to the regional lymph nodes, where the spores germinate and multiply.

Knaus will look at the interplay between anthrax toxins and lung epithelial cells—specialized cells that form a layer lining the lung. She is asking what happens to these cells when they are exposed to anthrax in the tiny airways of the lungs and how they are damaged by the toxins.

"It's really not clear at all what happens," says Knaus.

Bokoch will look at the effect of B. anthracis toxins on another type of innate immune cell, the neutrophil. Neutrophils, the most common white blood cell in circulation, are our first line of defense against invading pathogens. They search out bacteria and fungi and kill them with highly reactive oxidizing agents and toxic proteins. Bokoch and his colleagues have been studying these sorts of cells for many years.

"Because of the work that has been going on here in the Immunology Department at Scripps," says Bokoch, "we are really in a good position to identify these biochemical and genetic mechanisms."

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"It's clear that when someone is infected with Bacillus anthracis, [the bacterium] is able to suppress the immune system of that person," says Professor Gary Bokoch, who is one of the lead investigators on the TSRI grant. Photo by Jason S. Bardi.