Parallel evolution in Pseudomonas aeruginosa over 39,000 generations in vivo

Abstract

The Gram-negative bacterium Pseudomonas aeruginosa is a common cause of chronic airway infections in individuals with the heritable disease cystic fibrosis (CF). After prolonged colonization of the CF lung, P. aeruginosa becomes highly resistant to host clearance and antibiotic treatment; therefore, understanding how this bacterium evolves during chronic infection is important for identifying beneficial adaptations that could be targeted therapeutically. To identify potential adaptive traits of P. aeruginosa during chronic infection, we carried out global transcriptomic profiling of chronological clonal isolates obtained from 3 individuals with CF. Isolates were collected sequentially over periods ranging from 3 months to 8 years, representing up to 39,000 in vivo generations. We identified 24 genes that were commonly regulated by all 3 P. aeruginosa lineages, including several genes encoding traits previously shown to be important for in vivo growth. Our results reveal that parallel evolution occurs in the CF lung and that at least a proportion of the traits identified are beneficial for P. aeruginosa chronic colonization of the CF lung.

FIG 1

Pseudomonas aeruginosa isolates from three individuals with cystic fibrosis. Note that clonal groups Ca and Cb are isolated from the same individual, though they are different clonal groups (confirmed by RAPD assay and pulsed-field gel electrophoresis [data not shown]). More information is available in Table S1 in the supplemental material. SCV, small colony variant.

FIG 2

Hierarchical clustering of microarray data. Normalized raw microarray signals of 5,391 genes (mean values for two biological replicates) were used for clustering. We confirmed that clusters of each clonal group are not dependent on the similarity measurement method by using both a Spearman correlation coefficient (A) and Euclidean distance (B).

FIG 3

Microarray heat maps of genes differentially expressed between clonal groups. Differentially expressed genes between clonal group A (A), clonal group B (B), clonal group Ca (C), and clonal group Cb (D) are shown. Each heat map shows a subset of differentially expressed genes between clonal groups with signals of >1,000 in at least one strain. Comprehensive lists for these analyses are available in Table S4 in the supplemental material. Red indicates high levels of mRNA, and blue indicates low levels of mRNA.

FIG 4

Microarray heat map of differentially expressed genes within clonal groups. All strains in each clonal group were compared to their ancestor (initial strain) and evaluated for significant changes in gene expression (>2-fold change and FDR of <0.05). The numbers used to generate this heat map are the mean fold changes (log₂) in mRNA levels compared to the mRNA level of the ancestor strain of each clonal group. Red indicates an increase in the mRNA level, and blue indicates a decrease in the mRNA level. All genes detected in this analysis are reported separately in Table S6 in the supplemental material.

FIG 5

Common gene expression changes among three clonal groups. Venn diagram showing common changes in gene expression between three clonal groups (groups A, B, and Cb). The 24 genes commonly regulated in the three clonal groups are listed in Table 1. Genes that are positively regulated over time (+) and genes that are negatively regulated over time (-) are indicated (for instance, in the red circle at the top of the figure, of the 16 genes, 4 genes were positively regulated over time and 12 genes were negatively regulated over time.) The P value for the two clonal groups was calculated using a hypergeometric test and indicates that the probability of identifying the overlapping genes by random chance is very low. The full list of genes is available in Table S6 in the supplemental material.