Genetic and molecular evidence that MucA may be the sole determinant of mucoid conversion in Pseudomonas aeruginosa
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Date
2012-06
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The Ohio State University
Abstract
Cystic Fibrosis (CF) is the most common fatal hereditary disease among Caucasians, where chronic lung infections with the bacterium Pseudomonas aeruginosa are the leading cause of death. In CF, P. aeruginosa converts to a mucoid phenotype, causing a decline in patient survival. The most common mutations responsible for mucoidy disrupt a gene encoding the anti-sigma factor MucA. MucA-independent mechanisms contributing to mucoidy have been previously proposed; however, the gene(s) responsible remain uncharacterized. As a direct result, mucoid conversion of P. aeruginosa remains partially unclear. The aim of this work was to identify and characterize MucA independent mucoid conversion pathways. Along the way, we found strong evidence to suggest a MucA-independent pathway may not exist. First, the mucA gene was sequenced from a collection of laboratory-derived mucoid isolates. 20% were found to possess a wild type mucA and these isolates were chosen for further investigation into a MucA independent pathway. To confirm that MucA does not control alginate overexpression on a protein level, we overproduced MucA on an inducible plasmid. However, the mucoid phenotype of all isolates was complemented upon MucA overproduction, illustrating that we have yet to isolate mucoid colonies independent of MucA. To this end, we developed a plasmid-based screen for mucoid isolates whose phenotype cannot be complemented by MucA overproduction. Surprisingly, it remained that this screen yielded only mucoid derivatives with mutations in mucA. Despite increasing evidence that MucA is required for mucoid conversion, the previous studies suggest a MucA independent pathway exists, which is also independent of the sigma factor AlgT/U. Therefore, to elucidate this pathway we screened for mucoid isolates in an algT-deficient strain. Unfortunately, four independent mucoid conversion assays with and without sublethal hydrogen peroxide treatment resulted in no mucoid isolates. Concerned by these conflicting data, we acquired the original muc23 isolate previously published to be independent of MucA and determined that it too is complemented by MucA overexpression, demonstrating the importance of investigating post-transcriptional control of protein expression. These data suggest that MucA alterations are essential for mucoid conversion and may be the sole determinant of mucoid conversion in CF patients.
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Keywords
Pseudomonas, aeruginosa, mucoid, conversion, mucA