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Fibrosis: Finding the Way Forward to Treatment & Cure
CHORI Scientists Identify Pyocyanin As Key Player for Oxidative Stress and CFTR Impairment in Airways

"We hypothesized that pyocyanin would impair airway cell function by basically generating a corrosive environment, and by interfering with chloride ion transport."

Pyocyanin is a blue-green pigment and a small molecule virulence factor secreted by the bacterium, Pseudomonas aeruginosa. While common to many lung infections and immunocompromised patients, P. aeruginosa is also the major bacterium that colonizes the lungs of patients with cystic fibrosis (CF). However, no one has investigated the impact of pyocyanin, which in the lungs of CF patients can reach incredibly high concentrations, on function in CF cells. Until now, with a new publication by CHORI scientist, Beate Illek, PhD, in Free Radical Biology and Medicine.

Researchers have long known that pyocyanin generates reactive oxygen species by a mechanism called redox cycling, during which pyocyanin spontaneously transfers electrons to molecular oxygen.

As Dr. Illek explains, “The redox cycling of pyocyanin results in the release of superoxide radicals and the formation of hydrogen peroxide, or H202, a highly reactive oxygen species that leads to oxidative stress.”

Scientists have never investigated how pyocyanin’s redox cycling might impact the cystic fibrosis transmembrane conductance regular (CFTR ) chloride ion channel, however, the proper functioning of which is impaired in CF by a mutation in the CFTR gene. Under normal conditions, CFTR shuttles chloride and bicarbonate ions from one side of a cell membrane to the other side, which in turn, triggers the flow of water into the nasal passages, keeping mucus flowing.

“The mucus lining in lungs catches bacteria, viruses, dust and other inhaled particles, and the cilia then sweep that mucus out. But the level of hydration needs to be just right to keep the cilia beating which keeps that mucus flowing,” explains Dr. Illek.

Healthy lungs are able to respond to bacterial products by secreting chloride and fluid via the CFTR channel, which flushes the bacteria out that are trapped in the mucus.

“This is an important host defense mechanism that keeps our lungs sterile,” Dr. Illek says. “Because the Chloride channel is defective in CF patients, they don’t have the proper airway hydration to flush it out and all that sticky mucus leads to chronic lung infections.”

Many researchers, Dr. Illek included, hold out hope that targeted gene therapy to restore CFTR function can hold the key to treating CF. As a result, Dr. Illek, who recently received a research grant from the Cystic Fibrosis Foundation to investigate the role of pyocyanin-induced oxidative stress and metabolomics in CF Airways, specifically focused her pyocyanin research on CF cells in which the CFTR ion channel function had been restored.
“We wanted to mimic the conditions you would find in the clinic if you were treating patients with a CFTR rescue or gene therapy approach, in which CF patients would still be colonized with P. aerugi-nosa.”
“We wanted to ask the question, would it be possible to restore chloride ion transport in CF patients if they are fully colonized?” says Dr. Illek.

What Dr. Illek found was quite surprising and eye-opening: the major impact of pyocyanin is extremely damaging, resulting in near complete inhibition of chloride ion transport in CF cells that were CFTR corrected. In addition, the study revealed a dramatic impact of pyocyanin on cellular ATP levels, which are important for normal CFTR activity. The study also found that pyocyanin caused a severe depletion of glutathione which is an important antioxidant and might explain why glutathione levels are lower in CF patients.

“The important message here is that the inhibition of the CFTR channel may be an universal impact that pyocyanin has on all kinds of different CFTR activators that are in the drug discovery pipeline,” Dr. Illek explains.
While the results of Dr. Illek’s study highlight the continued challenges to the silver bullet approach, the race for a treatment or cure is far from over. The study identifies a critical limitation of CFTR therapy, but it also points the way forward for the development of alternative approaches to overcome this limitation.

“This suggests that we need to eradicate the Pseudomonas bacteria before CF therapy begins to activate the CFTR pathway, in particular to lower the pyocyanin levels currently found in CF patients,” says Dr. Illek. “The good news is that some medications have already been shown to do that, and we are currently searching for additional compounds that inhibit P. aeruginosa’s production of pyocyanin.”

That’s only one possible tack, however, as Dr. Illek explained at a recent invited discussion at the 22nd Annual North American Cystic Fibrosis Conference, 2008.. Others include developing a CFTR activator that is not sensitive to pyocyanin’s inhibitory effects, or identifying as of yet undiscovered cellular targets.

“We need to better understand the metabolic changes that occur in the epithelial cells so that we can identify novel cellular targets that could make the cells themselves resistant to pyocyanin,” says Dr. Illek.

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Tuesday, May 17, 2011 8:19 AM

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