Evolution of Pseudomonas aeruginosa in the Cystic Fibrosis Lung
Pseudomonas aeruginosa is the primary cause of chronic endobronchial infections in the CF-lung lung, where it encounters selective pressures that include iron-limitation, antibiotics and immune system surveillance. This pathogens extraordinary adaptability arises in part from a large and malleable genome that is rich in regulatory elements. Pseudomonas adaptive potential may also be increased by the activity of so-called mutator strains that have high background mutation rates. We sought to discover large- and fine-scale patterns of genomic changes in 49 strains representing 16 longitudinal series that ranged up to 25 months in duration. A subset of these contained mutators, operationally defined as producing RifR mutants at a frequency one order of magnitude or greater than the population median (2.5x10-8). Strains were phenotyped with respect to mucoid status, antibiotic resistances, and frequency of RifR mutants, and genotyped with respect to presence of plasmids, rep-PCR (BOXA1, ERIC) and PFGE (SpeI and DraI) fingerprints, and multi-locus sequence types (MLST) at acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE. Mutators were present in 6 of 16 series, and mutator clones were more likely to be multiply antibiotic resistant. Clone fingerprints within series were consistently more similar than those between series. Banding patterns of initial and terminal clones from each series were distinct from initial and terminal clones from all other series. Major architectural changes were confined to 6 series, all of which resulted in genome size reduction. There was no correlation between instances of architectural change and mutator status. 3 series contained isolates whose fingerprints were not shared by any clone in either the series or the collection which suggests co-infection with multiple strains.
A common environmental microbe Pseudomonas aeruginosa is the primary cause of terminal endobronchial infections in cystic fibrosis patients. This opportunistic pathogens extraordinary adaptability arises in part from a large and malleable genome that is rich in regulatory elements. The adaptive potential of Pseudomonas may also be increased by the activity of mutator strains. To investigate the evolutionary role played by mutators, and to identify gene deletions and amplifications that arise and persist in chronic infections we undertook DNA microarray-based comparative genome hybridization and CLONDIAG miniarray analyses on 21 clinical P. aeruginosa samples isolated from 5 CF patients. We used these data to test four hypotheses: (1) isolates within lineages evolve clonally; (2) mutator-containing lineages contain more deletions and amplifications than non-mutator lineages; (3) genes reported as being required for the onset of infection are deleted during the course of chronic infection; (4) genome size decreases overall during the course of chronic infection. Mutators were present in 3 of 5 patient series; no significant correlation was observed between mutator presence and the observed number of mutation events. A dendrogram generated from array profiles indicates clonal relationships between isolates within a patient series, but not among patients. However, population heterogeneity within patients is still evident by the non-cumulative nature of gene copy-number changes. Two large regions of sequential copy-number decreases are observed. These span 106 and 114 open reading frames, respectively, and contain genes involved in transcriptional regulation, amino acid transport and metabolism, cell wall and membrane modifications, metal transport and metabolism, toxin production and defense as well as general carbohydrate and cell-signaling pathway. Interestingly, these categories are also represented by 31 decreases in gene-copy number shared by at least one isolate across all 5 patient series. The majority of changes within these functional groups can be construed to decrease antigenicity. The spectrum of functionally redundant loci mutated suggest the Pseudomonas genome has multiple potential targets for selection that affect phenotypes required for persistence, of which a small portion are mutated in a given strain. These data support all but our second hypotheses as this collection revealed no association between mutator status and frequency of mutation events.
Advisor:R. Frank Rosenzweig; Scott Miller; Mark Pershouse
School:The University of Montana
School Location:USA - Montana
Source Type:Master's Thesis
Keywords:division of biological sciences
Date of Publication:08/07/2008