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Scientists discover how anthrax creates its deadly spores


Knowledge could lead to new vaccines, treatments, detection and decontamination technologies

In the age-old battle between man and microbe, it pays to know your enemy. This is especially true for Bacillus anthracis, the bacterium that causes anthrax. Tiny spores of this highly infectious pathogen can survive drought, bitter cold and other harsh conditions for decades, yet still germinate almost instantly to infect and kill once inside an animal or human host.

In a collaboration funded by the U.S. Office of Naval Research and the National Institutes of Health, scientists from three major research institutions - the University of Michigan, The Institute for Genomic Research (TIGR), and The Scripps Research Institute - are working together to identify the genes and proteins involved in anthrax’s deadly metamorphosis. Their work provides information other researchers can use to develop new vaccines and treatments targeted at specific points in the complex process of anthrax growth and spore formation.

The first results of the collaboration’s work will be published as the cover story in the Jan. 1, 2004 issue of the Journal of Bacteriology and posted Dec. 18, 2003 on the journal’s web site. This study is the first analysis of a bacterial pathogen using the combined investigative tools of genomics and proteomics. It is also the first study to document, at a molecular level, all the genes and proteins involved in B.anthracis spore formation.

Major findings of the study include:
  • When compared to other bacteria, anthrax spore formation is an unusually complex and intricate process.

  • Up to one-third of all the genes in the Bacillus anthracis genome are involved in spore production.

  • Genes are expressed in five discrete phases over a five-hour time period.

  • Each mature anthrax spore contains about 750 individual proteins.

"The most surprising result of this study is the degree of dedication this organism devotes to making its spore," says Philip C. Hanna, Ph.D., an assistant professor of microbiology and immunology in the U-M Medical School and the paper’s corresponding author. "It may require one-third of the entire genome. This shows how important the spore is to this organism’s life cycle. The spore allows the anthrax bacterium to survive conditions that would kill most other living things."

Using cutting-edge techniques of functional genomics and proteomics analysis, scientists in the collaboration were able to shed new light on the molecular biology of the anthrax spore," says Scott N. Peterson, Ph.D., one of several scientists from The Institute for Genomic Research (TIGR) in Rockville, MD who are co-authors on the paper.

"Until recently, we only knew how the anthrax spore was made on a microscopic level. We could see different structures forming, but didn’t know precisely what went into making them," says Nicholas H. Bergman, Ph.D., a research investigator in the U-M Bioinformatics Program and a primary author of the paper. "Now we have a much clearer view of how the spore is assembled, and exactly what it is made of."

Bacillus and Clostridium (the bacterium that causes tetanus) are the only bacteria that can shut down normal metabolic functions and convert rapidly into dormant, protective spores when environmental conditions make it impossible for them to otherwise survive.

Scientists have been studying anthrax spores since 1876, when they were first described by the pioneering German bacteriologist, Robert Koch. Like a golf ball, anthrax spores are made of many layers of material, which protect DNA in the core.

The spore’s tough outer coat is surrounded by a loose-fitting layer called the exosporium. When the spore gets inside a human or animal host - the first step in the infection process - sensing agents in the exosporium signal the spore to "hatch," or germinate, and start producing more bacteria.

TIGR scientists used DNA microarray technology to monitor gene expression changes in Bacillus anthracis over time as cells transitioned from growth to spore formation. "Since the spore is the infectious particle of the anthrax bacterium, it made sense to focus initially on the molecular biology of the spore," says Peterson.

Scientists at The Scripps Research Institute in La Jolla, CA used advanced proteomics analysis technologies to identify proteins expressed in anthrax spores. "Proteomics experiments can reveal the expression and localization of proteins in microorganisms," says John R. Yates, Ph.D., a professor of cell biology at Scripps Research. "This is important, because some of these proteins may be promising targets for future vaccine development."

Hongbin Liu, Ph.D., a former Scripps Research post-doctoral research fellow and the paper’s first author, adds that the study "clearly demonstrates the benefits of combining genomics and proteomics in a single study. The combined approach helped deepen our understanding of the complexity of spore growth and sporulation."

Microbiologists at the U-M Medical School were responsible for working with the bacteria to study how it infects and causes disease in its human host. U-M scientists worked with an attenuated strain of B. anthracis, which was modified to make it safe to handle in university laboratory facilities. U-M also provided the bioinformatics technology and expertise required to analyze the large amounts of data generated by the study.

The collaboration’s scientists identified 2,090 B. anthracis genes, of nearly 6,000 in the entire genome, which appear to be involved in spore formation. Gene activity occurred in five overlapping waves spread across a five-hour time period, but actual construction of the spore didn’t begin until the fourth wave of gene expression.

According to Hanna, this suggests that a surprising number of gene products in the spore itself are not produced during spore formation, but rather are scavenged from the vegetative bacillus during the process.

"Think of these proteins as the supplies required to build a house - like lumber, nails, and shingles," says Hanna. "Many of these proteins already exist in the bacillus and can be recycled to create the spore. The first step is not to produce them, but simply to collect them all in one place and then re-pack them into a spore. Other accessory genes contain instructions for making regulatory proteins and enzymes, which are tools the anthrax bacillus uses to construct its spore."

Data from the study indicates that proteins produced during the large, fifth wave of gene expression also become part of the spore itself. These include enzymes the spore needs for rapid germination, virulence factors and proteins that help the bacterial cell survive in a new host environment.

"When it enters the body, the spore has all the digestive enzymes it needs packed inside," explains Hanna. "Immediately after germination, the cell can start eating, multiplying and spreading throughout the body."

Complete data from the collaboration’s study of the genome and proteome of B. anthracis spores has been posted on the National Center for Biotechnology Information’s Gene Expression Omnibus database at, where it will be freely available to the scientific community. Hanna stresses that, while the data will be extremely valuable to biomedical researchers, it has no value related to the use of anthrax as a biological weapon.

"We want our results to be available to all university and corporate researchers developing anthrax spore countermeasures," says Hanna. "The scientific skills and technologies developed by the collaboration can now be focused on the next stage of anthrax infection - interaction with the host."

In the next phase of their research, collaboration scientists will examine changes in gene expression and protein synthesis that occur when the anthrax bacillus enters immune system cells in the host.

"The spore is the infectious agent of anthrax. It’s how the bacterium persists in the natural environment, and it’s what terrorists would manipulate in a bioterrorism attack," says Brendan Thomason, a U-M graduate student and a co-author of the paper. "In order to understand how the bacterium causes disease and discover new methods for anthrax treatment and prevention, scientists need a more thorough understanding of the intricacies of the spore."

Additional collaborators in the research study include: Shamira Shallom, Alyson Hazen, David A. Rasko, Ph.D., Jacques Ravel, Ph.D., and Timothy D. Read, Ph.D., from TIGR, and U-M research associate, Joseph Crossno.

Journal of Bacteriology, Vol.186, pp. 164-178, Jan. 2004

Contact: Sally Pobojewski,, (734) 615-6912 (U-M)
Robert Koenig,, (301) 838-5880 (TIGR)
Jason Bardi,, (858) 784-9254 (Scripps Research)

Sally Pobojewski | EurekAlert!
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