Within-host evolution of Pseudomonas aeruginosa reveals adaptation toward iron acquisition from hemoglobin

Abstract

ABSTRACT Pseudomonas aeruginosa airway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist, P. aeruginosa depends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and the Pseudomonas heme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation of P. aeruginosa to the host environment. Here we investigated the within-host evolution of the transmissible P. aeruginosa DK2 lineage. We found positive selection for promoter mutations leading to increased expression of the phu system. By mimicking conditions of the CF airways in vitro, we experimentally demonstrate that increased expression of phuR confers a growth advantage in the presence of hemoglobin, thus suggesting that P. aeruginosa evolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additional P. aeruginosa lineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages, phuR promoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment of P. aeruginosa infections in CF patients. IMPORTANCE Most bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens' ability to acquire iron. Here, we investigated the within-host evolution of P. aeruginosa, and we found evidence that P. aeruginosa during long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

FIG 1

Maximum-parsimony phylogenetic tree showing the genetic relationship of the 11 DK2 clones included in this study. The phylogenetic tree is a subset of a phylogenetic tree from the work of Marvig et al. (2), who recently reported the genome sequences of 55 DK2 isolates. The shown tree depicts the genetic relationship of the 11 DK2 isolates included in this study, and it represents a total of 1,827 mutations (1,486 SNPs and 311 insertion/deletions) identified from whole-genome sequencing. Lengths of branches are proportional to the numbers of mutations except in the case of the truncated branch leading to isolate DK2-CF222-2001. For this hypermutator isolate, the large number of mutations is indicated at the end of the truncated branch. We searched the genomes for nonsynonymous mutations within genes encoding components of the pyoverdine, pyochelin, phu, has, feo, and fec iron acquisition systems (7, 11–13), and circles on the evolutionary branches denote that the specified gene is mutated in the branch. Due to the large number of mutations in the branch leading to the hypermutable isolate DK2-CF222-2001, only phuR and phuSTUVW intergenic mutations are specified. *, in addition to the three phuR and phuSTUVW intergenic mutations, this branch also contains nonsynonymous mutations in pvdS, pvdL, fpvI, the FpvAII gene, fpvR, phuR, fptA, pchH, pchG, pchF, pchE, and pchD (2).

FIG 2

Overview of the intergenic region upstream of phuR. The alignment shows homologue sequences from different isolates with genetic variants highlighted in bold. Wild-type sequences of P. aeruginosa strains PAO1, DK1, DK2, and C are shown at the top of the alignment. Abbreviations of sequence alleles from different isolates are indicated in parentheses (WT and M1 to M10). Positions of promoters and a Fur box are indicated with black lines above the alignment (the phuSTUVW promoter is only partially shown). Positions are relative to the start codon of phuR.

FIG 3

Relative transcriptional levels of genes encoding the phu system. The transcriptomes of six of the DK2 isolates included in this study have previously been measured at exponential growth phase in LB medium (4). The expression of the phu genes is shown for each of the six clinical isolates relative to that for laboratory reference strain PAO1. Values are averages for three replicates, and the values are normalized relative to the transcription of the respective gene in strain PAO1.

FIG 4

Overview of pvdS mutations in the DK1 and C lineages. Mutations that have accumulated in evolved isolates relative to sequences of their ancestor are shown. The pvdS mutation found in the DK2 lineage is shown in Fig. 1.

FIG 5

Pyoverdine production in isolates of P. aeruginosa. The presence of pyoverdine secreted into the supernatant of bacterial cultures grown in pyoverdine-inducing medium was quantified by measurement of the absorbance at OD₄₀₅ and normalized against the cell density (OD₆₀₀). The means and standard deviations calculated from three biological replicates are shown in the bar plot.