Lars Jelsbak

Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective

The airways of patients with cystic fibrosis (CF) are nearly always infected with many different microorganisms. This environment offers warm, humid and nutrient-rich conditions, but is also stressful owing to frequent antibiotic therapy and the host immune response. Pseudomonas aeruginosa is commonly isolated from the airways of patients with CF, where it most often establishes chronic infections that usually persist for the rest of the lives of the patients. This bacterium is a major cause of mortality and morbidity and has therefore been studied intensely. Here, we discuss how P. aeruginosa evolves from a state of early, recurrent intermittent colonization of the airways of patients with CF to a chronic infection state, and how this process offers opportunities to study bacterial evolution in natural environments. We believe that such studies are valuable not only for our understanding of bacterial evolution but also for the future development of new therapeutic strategies to treat severe chronic infections.

Evolutionary dynamics of bacteria in a human host environment

Laboratory evolution experiments have led to important findings relating organism adaptation and genomic evolution. However, continuous monitoring of long-term evolution has been lacking for natural systems, limiting our understanding of these processes in situ. Here we characterize the evolutionary dynamics of a lineage of a clinically important opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it adapts to the airways of several individual cystic fibrosis patients over 200,000 bacterial generations, and provide estimates of mutation rates of bacteria in a natural environment. In contrast to predictions based on in vitro evolution experiments, we document limited diversification of the evolving lineage despite a highly structured and complex host environment. Notably, the lineage went through an initial period of rapid adaptation caused by a small number of mutations with pleiotropic effects, followed by a period of genetic drift with limited phenotypic change and a genomic signature of negative selection, suggesting that the evolving lineage has reached a major adaptive peak in the fitness landscape. This contrasts with previous findings of continued positive selection from long-term in vitro evolution experiments. The evolved phenotype of the infecting bacteria further suggests that the opportunistic pathogen has transitioned to become a primary pathogen for cystic fibrosis patients.

In Situ Growth Rates and Biofilm Development of Pseudomonas aeruginosa Populations in Chronic Lung Infections

The growth dynamics of bacterial pathogens within infected hosts are a fundamental but poorly understood feature of most infections. We have focused on the in situ distribution and growth characteristics of two prevailing and transmissible Pseudomonas aeruginosa clones that have caused chronic lung infections in cystic fibrosis (CF) patients for more than 20 years. We used fluorescence in situ hybridization (FISH) directly on sputum specimens to examine the spatial distribution of the infecting P. aeruginosa cells. Mucoid variants were present in sputum as cell clusters surrounded by an extracellular matrix, whereas nonmucoid variants were present mainly as dispersed cells. To obtain estimates of the growth rates of P. aeruginosa in CF lungs, we used quantitative FISH to indirectly measure growth rates of bacteria in sputum samples (reflecting the in vivo lung conditions). The concentration of rRNA in bacteria isolated from sputa was measured and correlated with the rRNA contents of the same bacteria growing in vitro at defined rates. The results showed that most cells were actively growing with doubling times of between 100 and 200 min, with some growing even faster. Only a small stationary-phase subpopulation seemed to be present in sputa. This was found for both mucoid and nonmucoid variants despite their different organizations in sputum. The results suggest that the bacterial population may be confronted with selection forces that favor optimized growth activities. This scenario constitutes a new perspective on the adaptation and evolution of P. aeruginosa during chronic infections in CF patients in particular and on long-term infections in general.

Evolution and diversification of Pseudomonas aeruginosa in the paranasal sinuses of cystic fibrosis children have implications for chronic lung infection

The opportunistic pathogen Pseudomonas aeruginosa is a frequent colonizer of the airways of patients suffering from cystic fibrosis (CF). Depending on early treatment regimens, the colonization will, with high probability, develop into chronic infections sooner or later, and it is important to establish under which conditions the switch to chronic infection takes place. In association with a recently established sinus surgery treatment program for CF patients at the Copenhagen CF Center, colonization of the paranasal sinuses with P. aeruginosa has been investigated, paralleled by sampling of sputum from the same patients. On the basis of genotyping and phenotypic characterization including transcription profiling, the diversity of the P. aeruginosa populations in the sinuses and the lower airways was investigated and compared. The observations made from several children show that the paranasal sinuses constitute an important niche for the colonizing bacteria in many patients. The paranasal sinuses often harbor distinct bacterial subpopulations, and in the early colonization phases there seems to be a migration from the sinuses to the lower airways, suggesting that independent adaptation and evolution take place in the sinuses. Importantly, before the onset of chronic lung infection, lineages with mutations conferring a large fitness benefit in CF airways such as mucA and lasR as well as small colony variants and antibiotic-resistant clones are part of the sinus populations. Thus, the paranasal sinuses potentially constitute a protected niche of adapted clones of P. aeruginosa, which can intermittently seed the lungs and pave the way for subsequent chronic lung infections.

Early adaptive developments of Pseudomonas aeruginosa after the transition from life in the environment to persistent colonization in the airways of human cystic fibrosis hosts

Pseudomonas aeruginosa is an opportunistic pathogen ubiquitous to the natural environment but with the capability of moving to the host environment. Long-term infection of the airways of cystic fibrosis patients is associated with extensive genetic adaptation of P. aeruginosa, and we have studied cases of the initial stages of infection in order to characterize the early adaptive processes in the colonizing bacteria. A combination of global gene expression analysis and phenotypic characterization of longitudinal isolates from cystic fibrosis patients revealed well-known characteristics such as conversion to a mucoid phenotype by mucA mutation and increased antibiotic resistance by nfxB mutation. Additionally, upregulation of the atu operon leading to enhanced growth on leucine provides a possible example of metabolic optimization. A detailed investigation of the mucoid phenotype uncovered profound pleiotropic effects on gene expression including reduction of virulence factors and the Rhl quorum sensing system. Accordingly, mucoid isolates displayed a general reduction of virulence in the Caenorhabditis elegans infection model, altogether suggesting that the adaptive success of the mucoid variant extends beyond the benefits of alginate overproduction. In the overall perspective the global phenotype of the adapted variants appears to place them on paths in direction of fully adapted strains residing in long-term chronically infected patients.

Pattern formation by a cell surface-associated morphogen in Myxococcus xanthus

In response to starvation, an unstructured population of identical Myxococcus xanthus cells rearranges into an asymmetric, stable pattern of multicellular fruiting bodies. Central to this pattern formation process are changes in organized cell movements from swarming to aggregation. Aggregation is induced by the cell surface-associated C-signal. To understand how aggregation is accomplished, we have analyzed how C-signal modulates cell behavior. We show that C-signal induces a motility response that includes increases in transient gliding speeds and in the duration of gliding intervals and decreases in stop and reversal frequencies. This response results in a switch in cell behavior from an oscillatory to a unidirectional type of behavior in which the net-distance traveled by a cell per minute is increased. We propose that the C-signal-dependent regulation of the reversal frequency is essential for aggregation and that the remaining C-signal-dependent changes in motility parameters contribute to aggregation by increasing the net-distance traveled by starving cells per minute. In our model for symmetry-breaking and aggregation, C-signal transmission is a local event involving direct contacts between cells that results in a global organization of cells. This pattern formation mechanism does not require a diffusible substance or other actions at a distance. Rather it depends on contact-induced changes in motility behavior to direct cells appropriately

Microbial ecology and adaptation in cystic fibrosis airways

Chronic infections in the respiratory tracts of cystic fibrosis (CF) patients are important to investigate, both from medical and from fundamental ecological points of view. Cystic fibrosis respiratory tracts can be described as natural environments harbouring persisting microbial communities with Pseudomonas aeruginosa as a dominant pathogen. Various factors contribute to the complexity of this ecosystem, including community composition, dynamics and interactions, as well as heterogeneous distribution and fluctuation of components of the immune system, antibiotics and nutrients. All these elements constitute the selective forces that drive the evolution of the microbes after they migrate from the outer environment to human airways. Pseudomonas aeruginosa adapts to the new environment through genetic changes and exhibits a special lifestyle in chronic CF airways. Understanding the persistent colonization of microbial pathogens in CF patients in the context of ecology and evolution will expand our knowledge of the pathogenesis of chronic infections and improve therapeutic strategies.

Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts

The genetic basis of bacterial adaptation to a natural environment has been investigated in a highly successful Pseudomonas aeruginosa lineage (DK2) that evolved within the airways of patients with cystic fibrosis (CF) for more than 35 y. During evolution in the CF airways, the DK2 lineage underwent substantial phenotypic changes, which correlated with temporal fixation of specific mutations in the genes mucA (frame-shift), algT (substitution), rpoN (substitution), lasR (deletion), and rpoD (in-frame deletion), all encoding regulators of large gene networks. To clarify the consequences of these genetic changes, we moved the specific mutations, alone and in combination, to the genome of the reference strain PAO1. The phenotypes of the engineered PAO1 derivatives showed striking similarities with phenotypes observed among the DK2 isolates. The phenotypes observed in the DK2 isolates and PAO1 mutants were the results of individual, additive and epistatic effects of the regulatory mutations. The mutations fixed in the σ factor encoding genes algT, rpoN, and rpoD caused minor changes in σ factor activity, resulting in remodeling of the regulatory networks to facilitate generation of unexpected phenotypes. Our results suggest that adaptation to a highly selective environment, such as the CF airways, is a highly dynamic and complex process, which involves continuous optimization of existing regulatory networks to match the fluctuations in the environment.

Bacterial adaptation during chronic infection revealed by independent component analysis of transcriptomic data

BackgroundBacteria employ a variety of adaptation strategies during the course of chronic infections. Understanding bacterial adaptation can facilitate the identification of novel drug targets for better treatment of infectious diseases. Transcriptome profiling is a comprehensive and high-throughput approach for characterization of bacterial clinical isolates from infections. However, exploitation of the complex, noisy and high-dimensional transcriptomic dataset is difficult and often hindered by low statistical power.ResultsIn this study, we have applied two kinds of unsupervised analysis methods, principle component analysis (PCA) and independent component analysis (ICA), to extract and characterize the most informative features from transcriptomic dataset generated from cystic fibrosis (CF) Pseudomonas aeruginosa isolates. ICA was shown to be able to efficiently extract biological meaningful features from the transcriptomic dataset and improve clustering patterns of CF isolates. Decomposition of the transcriptomic dataset by ICA also facilitates gene identification and gene ontology enrichment.ConclusionsOur results show that P. aeruginosa employs multiple patient-specific adaption strategies during the early stage infections while certain essential adaptations are evolved in parallel during the chronic infections.