The effect of a temperature-sensitive prophage on the evolution of virulence in an opportunistic bacterial pathogen

Abstract

Viruses are key actors of ecosystems and have major impacts on global biogeochemical cycles. Prophages deserve particular attention as they are ubiquitous in bacterial genomes and can enter a lytic cycle when triggered by environmental conditions. We explored how temperature affects the interactions between prophages and other biological levels using an opportunistic pathogen, the bacterium Serratia marcescens, which harbours several prophages and that had undergone an evolution experiment under several temperature regimes. We found that the release of one of the prophages was temperature-sensitive and malleable to evolutionary changes. We further discovered that the virulence of the bacterium in an insect model also evolved and was positively correlated with phage release rates. We determined through analysis of genetic and epigenetic data that changes in the bacterial outer cell wall structure possibly explain this phenomenon. We hypothezise that the temperature-dependent phage release rate acted as a selection pressure on S. marcescens and that it resulted in modified bacterial virulence in the insect host. Our study system illustrates how viruses can mediate the influence of abiotic environmental changes to other biological levels and thus be involved in ecosystem feedback loops.

Keywords: epigenetics; experimental evolution; opportunistic pathogen; prophage induction.

FIGURE 1

Overview of the relationships between our study and previous studies based on the same evolution experiment (Bruneaux et al., ; Ketola et al., 2013). Boxes are colour‐coded based on which study each item originates from.

FIGURE 2

Effect of evolutionary treatment and assay temperatures on the release rates of prophage PP4. Assays lasted 2 days and assay temperatures are given as day1/day2. (a) Posteriors of the model‐estimated mean for each treatment/assay combination. Points are estimated phage release rates for each of the 29 sequenced clones. Full posterior estimates for individual strains are shown in Figure S5. (b) Estimates of the assay temperature effects and (c) estimates of the evolutionary treatment effects, with the reference strain used as a reference point. Posteriors are shown as median and 95% credible interval. One‐sided Bayesian p‐values for pairwise comparisons denoted by * (p < 0.05) and *** (p < 0.001).

FIGURE 3

Putative PP4 phage particles observed in transmission electron microscopy (TEM). Negative staining with phosphotungstic acid (PTA) was used. All panels are shown to the same scale.

FIGURE 4

Effect of evolutionary treatment on strains virulence in waxmoth larvae at two incubation temperatures. (a) Relative virulence of individual sequenced clones, measured as relative hazards estimated from a Bayesian implementation of a Cox proportional‐hazards model. All virulence estimates are relative to the virulence of the reference strain in incubation at 24°C (denoted by a broken horizontal line) and are corrected for the effects of injection batch, larval body mass and optical density of injected cultures. (b) Mean relative virulence per evolutionary treatment and per incubation temperature as estimated by the model (exp(μ _evo)) (n = 29 sequenced clones). (c) Confirmatory results from a similar virulence experiment utilizing more bacterial clones from the same original evolution experiment (n = 222 clones, incubation at room temperature (RT) and at 36°C, virulence relative to the average virulence of the clones evolved at 31°C when incubated at room temperature). For each model parameter, 95% credible interval and median of the posterior are shown.

FIGURE 5

Relationship between relative bacterial virulence in waxmoth larvae and phage release rate for the sequenced evolved strains used in this study (n = 28) and the reference strain. Each data point represents one strain at a given assay temperature (24°C: data from virulence assay at 24°C and phage release assay at 24/24°C; 31°C: data from virulence assay at 31°C and phage release assay at 31/31°C). Bacterial virulence is relative to the reference strain in incubation at 24°C, similarly to Figure 4. Trend lines within each assay temperature are added for visual support only and are built using ordinary least squares regressions (95%‐enveloppes built using 500 bootstraps).

FIGURE 6

Alignment of the genomes from the 29 sequenced strains showing the variable genetic loci. Each circular track represents a sequenced genome, for which the evolutionary treatment is colour‐coded. Minor alleles for genetic variants are shown on the genome tracks in light grey (SNPs) and dark grey (indels). Ticks outside the last genome track indicate nonsynonymous variants (i.e. nonsynonymous SNPs and indels resulting in a frame shift). The outer line represents coordinates along the genome and the locations of the five predicted prophages. Prophages PP4 and PP7 are the prophages shown in Figure S4.

FIGURE 7

Association between phage release, virulence, and genetic variants observed in at least two sequenced strains. (a) Distribution of genetic variants across evolutionary treatments and association between alleles and phenotypes based on Wilcoxon rank sum tests. Variant IDs can be matched with those in Table S3 for details. (b) Visualization of the association between phenotypic values and major (M) and minor (m) alleles of variant a and of the “pooled” variants for galactokinase and glycosyltransferase. Colours correspond to the evolutionary treatment applied to each strain.

FIGURE 8

Association between phage release, virulence, and adenine methylation changes. The heatmap shows Spearman's ρ between methylated fractions of variable m6A epiloci (rows) and phenotypes (columns; Ph., phage release; Vir., bacteria virulence in waxmoth larvae). Overlapping or closest (≤500 bp) downstream genes were assigned to each m6A epiloci. Probable gene functions were assigned to each gene product based on a manual literature search. Visualization of the relationship between m6A methylated fractions and phenotypic trait values is shown for the heatmap cells marked with a letter. The m6A epiloci used in this figure were the variable m6A epiloci with a methylation fraction range ≥0.2 across sequenced samples and an uncorrected p‐value ≤0.005 for Spearman's ρ with at least one phenotypic trait.