Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages

Abstract

The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.

Fig 1. Evolutionary dynamics of the populations evolved within macrophages.

Dynamics of frequency of neutral marker in the 20 replicate evolving lines (in (A) lines A to J; in (B) lines K to T) and variation in population size along the evolution experiment (in (C) lines A to J; in (D) lines K to T).

Fig 2. Fitness measures of evolved clones.

In blue, the competitive fitness of evolved populations in the presence of MΦ: evolved clones (a sample of 30 from each indicated population) were competed against the ancestral clone (1:1). In orange, competitive fitness assay in the absence of MΦ. Error bars correspond to 2SE.

Fig 3. Genomic maps of the mutations detected in the sequenced evolved clones.

Red inverted triangles represent insertions of IS elements, blue lines mark single nucleotide polymorphisms and green triangles denote small insertions (pointing downwards) or deletions (pointing upwards). All events have either the aminoacid changes associated (for SNPs), the IS element inserted or the small number of base pairs deleted or inserted.

Fig 4. Invasion of an adaptive mutation at the rfaI locus.

The blue line shows the selective sweep leading to the fixation of an IS5 insertion likely causing gene loss of function. In the inset an estimate of the selection coefficient (s) of this mutation is given. s is the slope of the logarithm of the ratio between the number of clones carrying the mutation and those with the wild-type allele.

Fig 5. Bacterial survival under iron and or oxidative stress.

Log₁₀(Number of bacteria after 1h)- Log₁₀ (Number bacteria at 0h) in the environments: (A) Effect of Fe³⁺ depletion tested in minimal media supplemented with the indicated concentrations of Fe³⁺ and Ferrichrome; no significant difference between evolved and ancestral clones could be detected (T-test, P = 0.8 and P = 0.6, n = 5) (B) Effect of H₂O₂ stress tested in minimal media supplemented 2mM of H₂O₂; no significant difference between evolved and ancestral clones (T-test, P = 0.8, n = 10). (C) Fitness advantage of evolved clone is detected under both selective pressures, i.e. minimal media supplemented with the Fe³⁺, Ferrichrome and 2mM of H₂O₂ (T-test, P = 0.016, P = 0.02, n = 5)

Fig 6. Growth curves of evolved populations and the ancestral strain in minimal media with maltose or glucose (0.4%).

Each color on the growth curves represents similar patterns amongst the populations.