Editor's note: Despite receiving an excellent score in the highly competitive NIH funding process, Dr. Schiller's grant proposal (entitled "Characterization of AlgL and its Role in Alginate Synthesis in Pseudomonas aeruginosa") fell short of a funding award. CFRI awarded Dr. Schiller a grant so that he could continue his vital examination of Pseudomonas aeruginosa. The colonization of this bacteria in the lungs and our inability to control it results in severe pulmonary degeneration and death in many patients with CF. Dr. Neal Schiller is the chairman of the Graduate Program in Microbiology and the Associate Dean of the Graduate Division at the University of California at Riverside.
Pulmonary infections with the bacterium Pseudomonas aeruginosa remain intractable to antibiotic treatment, making these infections one of the most serious concerns of the cystic fibrosis patient. While recent investigations on the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) provide hope for the potential of gene therapy for CF, these strategies are unlikely to be effective in controlling or eradicating P. aeruginosa once established in the lung. My colleagues and I, as well as other investigators, have determined that the persistence of these bacteria in the face of antibiotic treatment and pulmonary defenses is attributable to the bacteria's ability to produce a protective exopolysaccharide coat, called alginate. Besides protecting the bacteria it surrounds, this high-molecular-weight polymer of mannuronic and guluronic acids is extremely viscous and is a contributing component of the thick mucous secretions that clog the patients' airways. Our approach to resolving these problems is to design strategies that either block alginate synthesis, degrade alginate off the surface of these bacteria, or degrade the free alginate in the lung. Funds provided by CFRI will be used to a) purify and characterize an enzyme that can degrade the alginate coat, and b) further characterize the bacterial genes involved in alginate synthesis in P. aeruginosa.
Several years ago, my colleagues and I were the first to clone and sequence the P. aeruginosa gene which encodes the protein alginate lyase, an enzyme that depolymerizes the alginate polymer. During these studies we made an important observation. The alginate lyase gene (called algL) is located on the bacterial chromosome within a cluster of genes involved in alginate biosynthesis. Moreover, all of the genes in this cluster appear to be co-regulated, that is, when the first one of these genes is turned on, they all are. This raised the rather paradoxical question: why would these bacteria arrange their genes in such a way that a degradative enzyme would be activated at the same time as the biosynthetic ones?
Our recent studies suggest that this arrangement is not accidental. It is our current working hypothesis that alginate lyase is actually required for alginate production, although exactly how this occurs remains speculative. The purification and characterization of the alginate lyase enzyme should answer this question. While many of the enzymes involved in alginate biosynthesis have been identified, several key components, including the polymerase and transport proteins, remain elusive. One of our goals is to determine all of the genes and enzymes needed for alginate synthesis. In so doing, we might discover novel ways to block the production of alginate by these bacteria. Alternately, by further studying and characterizing the enzyme alginate lyase, we hope to someday be able to use this enzyme to degrade the alginate layer off of these bacteria and depolymerize the alginate polymer in the CF patient's lungs. We recognize the difficulties with this therapeutic concept and the potential danger of initiating a harmful inflammatory response by introducing a foreign protein into a patient's lungs. Nevertheless, if we could devise a strategy whereby these bacteria and the lung secretions could be rendered alginate-free, then antibiotics and the patient's own pulmonary clearance mechanisms would be more effective against these microorganisms. Our hope is that a clearer understanding of the genes and enzymes involved in alginate production will stimulate novel ideas that can lead to the successful treatment and eradication of these bacteria.
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