Higher phage virulence accelerates the evolution of host resistance

Abstract

Pathogens vary strikingly in their virulence and the selection they impose on their hosts. While the evolution of different virulence levels is well studied, the evolution of host resistance in response to different virulence levels is less understood and, at present, mainly based on observations and theoretical predictions with few experimental tests. Increased virulence can increase selection for host resistance evolution if the benefits of avoiding infection outweigh resistance costs. To test this, we experimentally evolved the bacterium Vibrio alginolyticus in the presence of two variants of a filamentous phage that differ in their virulence. The bacterial host exhibited two alternative defence strategies: (1) super infection exclusion (SIE), whereby phage-infected cells were immune to subsequent infection at the cost of reduced growth, and (2) surface receptor mutations (SRM), providing resistance to infection by preventing phage attachment. While SIE emerged rapidly against both phages, SRM evolved faster against the high- than the low-virulence phage. Using a mathematical model of our system, we show that increasing virulence strengthens selection for SRM owing to the higher costs of infection suffered by SIE immune hosts. Thus, by accelerating the evolution of host resistance, more virulent phages caused shorter epidemics.

Keywords: experimental evolution; filamentous phages; resistance evolution; virulence.

Figure 1.

Population dynamics over 30 transfers. (a) Bacteria in CFU ml^–1. (b) Phages in PFU ml^–1; the horizontal grey dashed line represents the quantification limit below which quantifying filamentous phages using spectrophotometry is inaccurat. Note: free phages in the control treatment stem from the low-replicating resident phage VALGΦ6 (table 1). (c) Fraction of susceptible clones (%; n = 24). (d) Fraction of SIE hosts within phage-resistant clones. Fractions are based on 24 random clones per replicate population per timepoint. In all panels, data are represented as means of six replicate populations per treatment; error bars represent standard errors. Colours correspond to one of three experimental treatments: lower-virulence VALGΦ8_K04M1 (light red, dashed), higher-virulence VALGΦ8_K04M5 (dark red) and no phage (grey, dashed-dotted). (Online version in colour.)

Figure 2.

Phage prevalence (a) and fitness effects of evolved phage resistance versus immunity (b–e): (a) Phage prevalence for each co-evolving population in the presence of higher-virulence phage VALGΦ8_K04M5 (dark red) or the lower-virulence phage VALGΦ8_K04M1 (light red) over 30 transfers. (b) Darwinian fitness of SIE relative to SRM hosts. A value of unity corresponds to equal fitness. To account for potential costs associated with the GFP protein, competitions were performed where either the SIE or the SRM host was labelled (n = 3). (c) Correlation between bacterial growth rate [µ] and production of free phages measured as PFU ml^–1 per clone. (d) Phage particle production [PFU ml^–1] and (e) growth rate µ: both measured after 24 h of bacterial growth for SIE hosts, SRM hosts, clones from the control populations (grey) and the ancestral K01M1 strain (dotted horizontal line). Clones exposed to lower virulent VALGΦ8_K04M1 are shown in light red, clones exposed to higher virulent VALGΦ8_K04M5 in dark red. Phages from the ancestral K01M1, from SRM hosts and the control clones stem from an ancestral filamentous Vibrio phage VALGΦ6 integrated on chromosome 2 of K01M1 (table 1). Shown are means ± s.e., n = 24. (Online version in colour.)

Figure 3.

(a) Genetic loci on chromosome 1 under positive selection as indicated by parallel genomic evolution in populations exposed to phages: right: SIE hosts; middle: SRM hosts; left: zoom into MSHA-operon region from SRM hosts. Only loci that are not present in control populations are shown. Concentric circles correspond to one clone isolated from either the higher-virulence VALGΦ8_K04M5 (six inner circles, dark red) or the lower-virulence VALGΦ8_K04M1 phage (six outer circles, light red). Each small dot corresponds to one mutation event on the respective clone. HP, hypothetical protein; HP3 corresponds to locus tag K01M1_28150. For more detailed information on the underlying mutation, see electronic supplementary material, table S1. (b) Structure of the MSHA-operon and comparative genomics comprising MSHA-operons from V. alginolyticus FA2 (top), V. alginolyticus K01M1 and V. cholerae El Tor (bottom). Similarity between regions is indicated by dark grey blocks, genes with detected mutations are marked in orange, detected mutations are marked as arrows below V. alginolyticus K01M1. (Online version in colour.)

Figure 4.

Results of model simulations of 14 transfers for (a) bacteria in CFU ml^–1, (b) phages in PFU ml^–1, (c) SIE hosts and (d) SRM hosts depending on phage virulence (colour coded from blue: no virulence to red: high virulence). (Online version in colour.)