Oct. 3, 2003 A study by University of California, San Diego biochemists explains why infections of Pseudomonas bacteria, which affect 200,000 hospitalized patients each year in the United States, can be so dangerous to cells within the body, and points to new ways to treat those infections.
Pseudomonas aeruginosa, a common bacterium, can infect nearly every part of the body and produces toxins that damage tissues. In the study to be published in the October 17th issue of the Journal of Biological Chemistry, the researchers report that when the bacterium injects a toxin called "ExoU" with a tiny needle-like structure into cells, the toxin degrades phospholipids-greasy molecules that are a key component of cell membranes. They also found that chemicals known to block proteins that degrade phospholipids could save cells that would otherwise die. An early on-line version of the paper is available at http://www.jbc.org/cgi/reprint/M302472200v2.
"We have found that the toxin, which is associated with 90 percent of the severe cases of Pseudomonas infections, kills cells by targeting a component of the cell membrane," says Partho Ghosh, a professor of chemistry and biochemistry in UCSD's Division of Physical Sciences who headed the research team. "We have been able to identify chemicals that protect cells from the effects of the toxin, raising the possibility of a novel mode of treatment for these infections."
P. aeruginosa are widespread and, while these bacteria rarely affect healthy people, they are a serious problem for cystic fibrosis, AIDS, burn and chemotherapy patients and others with weakened immune systems. For example, 50 percent of deaths from AIDS are associated with P. aeruginosa infections and these bacteria are the leading cause of pneumonia contracted in intensive care units.
Furthermore, treating these infections is often problematic due to the antibiotic resistance of the bacteria. So the researchers wanted to better understand how the toxin killed cells in the hope that such insight would lead to alternative treatments for the infections.
"We knew that these bacteria use a needle-like structure to inject toxin directly into mammalian cells," explains Ghosh. "But we didn't know the mechanism by which the toxin induced cell death."
Three of their findings suggested that the ExoU toxin degrades cell membrane phospholipids. There were key similarities in the sequence of amino acids-building blocks that make up proteins-between the toxin and proteins known to degrade phospholipids. In addition, by attaching a fluorescent "tag" to the toxin, the researchers were able to see that ExoU injected into the cell ends up at the cell membrane. Finally, they found that chemical inhibitors of phospholipid degrading proteins protected cells from the toxin.
However, they also discovered that ExoU is not able to degrade phospholipids on its own, and that within cells, the toxin localizes to distinct regions of the inner cell membrane. Since phospholipids are spread throughout the cell membrane, this observation is consistent with the idea that the toxin is interacting with a specific factor, other than phospholipids, at the cell membrane.
"These results suggest that the toxin is inactive until it enters into mammalian cells, where it becomes activated through interaction with one or more host cell factors," explains Ghosh.
"We are currently looking for this activator in mammalian cells," says Rebecca Phillips, graduate student in the Ghosh lab and the first author on the paper. "It may be possible to find a drug which prevents this interaction and protects cells from the toxin."
The researchers are looking for multiple alternatives to treating the infection because it is still not clear that inhibitors of phospholipid degrading proteins can successfully treat ill patients, even if these chemicals seemed promising in the isolated mammalian cells the researchers used in their studies. This is because mammalian cells have their own proteins responsible for degrading phospholipids, which are needed for normal cell maintenance and repair. However, the researchers remain optimistic.
"It is definitely a possibility that we can find chemicals that are specific for the toxin and won't affect mammalian cells," says Phillips. "We are currently working to determine more detailed information about the molecular structure of the toxin, which will be useful in designing more specific drugs."
Other contributors to this work included Edward Dennis, a chemistry and biochemistry professor in UCSD's Division of Physical Sciences and UCSD's School of Medicine and David Six, a former graduate student in the Dennis lab. The research project was supported by the Cystic Fibrosis Foundation, National Institutes of Health and a Keck Distinguished Young Scholar in Medicine Award.
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