Friday, February 22, 2008

2nd Day-10th Talk: “Inhibiting Anthrax Spore Germination” by Ernesto Santos, Ph. D.

Dr. Santos joins us from the University of Nevada Las Vegas where he is a professor in the Department of Chemistry. His discussion today pertained to the germination of anthrax spores inside host macrophages.

Most bacteria will grow in the body and continue to divide and multiply exponentially; however anthrax has a mechanism that recognizes when it does not have enough food to survive. Under these nutrient poor conditions, this organism will develop spores. These spores are not anthrax (not even a bacterium at all), but have everything they need to become a bacterium upon germination. These spores are not fragile, they are resistant, and they can resist being heated with chloride (bleach) or radiation. They don’t need oxygen or food to survive and can stay dormant for years. These spores are interesting because they are resistant and dormant; however, as soon they reach a rich environment, they will germinate. Once the spores are taken up by mammalian cells, they are engulfed by macrophages in the immune system. Macrophages destroy everything, but anthrax has developed a stealth mechanism that allows it to germinate inside of the macrophages while withstanding harsh the harsh conditions of the phagolysosome. Once it germinates, the anthrax bacillus will produce toxins. Antibiotics can be used to kill the anthrax, but once it produces toxins, these toxins remain even once the cells have been destroyed.

Although spores are inert, they have a series of proteins in the membrane with sensors that surround the spore and detect small molecules (inosine and alanine). The first step in the research of these organisms was to determine the mechanism by which the spore detects these molecules, and this is done by studying the kinetics (changes in concentrations of these molecules over time). The spores are easy to follow when they germinate because they are dense (no water inside) and when they are irradiated with light, they will greatly defract it. Once the spores germinate, they swell and defraction goes down.

What happens once the spore binds these small molecules? This is still a black box. Once something binds, germination occurs, but we don’t know the physiologic processes by which this occurs. Both of these molecules (alanine and inosine) are needed for germination. If only one of these molecules is present, the spore will not germinate One hypothesis that Dr. Santos provided for why this may be is that the presence of alanine (an amino acid) and inosine (a nucleoside) infers that DNA and proteins are being produced. These factors would be significant evidence that the host is alive and will provide a nutrient rich environment for the spore to germinate. The more food there is available, the more rapid the germination.
Instead of doing kinetics on a single protein, they studies were performed on the whole organism. The general findings were: the more inosine you have, the faster the kinetics. Based on the slope of the line, there was more germination occurring. Two parameters obtained from the graph: Vmax and Km. Dr. Santos concluded there is a synergy between inosine binding and alanine concentration. As the alanine concentration is increased, inosine binding becomes tighter.
How does a spore bind alanine or inosine? Hypothetically, spores can bind inosine or alanine and then bind the other one or they can bind sequentially. Research has shown that there is a sequential order of events: sporeinosine binding alanine bindinggermination. The spore must germinate inside the macrophage to trigger germination. Current research is working on finding something that will bind to the spore without triggering germination. Competitive inhibitors of inosine were tested and it was found that if we compete against alanine, we end up having noncompetitive binding of inosine. It was predicted that the inhibitors are substrates that can bind to the same enzymes as inosine.

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