Balance between promiscuity and specificity in phage λ host range

Abstract

As hosts acquire resistance to viruses, viruses must overcome that resistance to re-establish infectivity, or go extinct. Despite the significant hurdles associated with adapting to a resistant host, viruses are evolutionarily successful and maintain stable coevolutionary relationships with their hosts. To investigate the factors underlying how pathogens adapt to their hosts, we performed a deep mutational scan of the region of the λ tail fiber tip protein that mediates contact with the receptor on λ's host, Escherichia coli. Phages harboring amino acid substitutions were subjected to selection for infectivity on wild type E. coli, revealing a highly restrictive fitness landscape, in which most substitutions completely abrogate function. A subset of positions that are tolerant of mutation in this assay, but diverse over evolutionary time, are associated with host range expansion. Imposing selection for phage infectivity on three λ-resistant hosts, each harboring a different missense mutation in the λ receptor, reveals hundreds of adaptive variants in λ. We distinguish λ variants that confer promiscuity, a general ability to overcome host resistance, from those that drive host-specific infectivity. Both processes may be important in driving adaptation to a novel host.

Fig. 1. A sequencing-based method to assess phage infectivity across thousands of variants.

a The experimental strategy. A library of phage variants was constructed and subjected to selection for the ability to infect E. coli. After each infectious cycle, barcodes corresponding to each variant were deeply sequenced, and their frequencies were used to score each variant using Model-Bounded Scoring. b Binding of λ to the host is mediated by the J protein, which contacts the receptor, LamB. J spontaneously forms a homotrimer and then associates with accessory proteins that fold J into a mature conformation [44]. c J is the 3′-most gene on a long polycistronic transcript expressed in late lytic phase that contains most of the capsid genes. We mutagenized the 3′-most 450 bp of J (excluding the stop codon) using NNN codon replacement and inserted a 15 bp barcode downstream of the gene. d For each of four missense variants at A94, five randomly selected barcodes are plotted by their abundance in the phage population after each infectious cycle relative to the pre-packaging DNA pool. Infectious cycle = 0 corresponds to the packaged but not yet selected phage population. Error bars represent the standard error between three replicate selections. For each variant, the growth rate, r, is the slope of an ordinary least squares regression line calculated separately for each barcode and averaged across all barcodes representing the same protein-level variant. The average growth rates for the selected variants are shown by slopes of the dashed lines. e Progeny per infectious cycle, equal to the exponential of the growth rate (e^r), is shown for λ bearing synonymous (to wild type) variants in orange, nonsense variants in purple, and single missense variants in mauve. Progeny is always smaller than the theoretical burst size for a given variant because progeny is dependent on the probability of a virion binding during the binding step (~0.5 for wt λ) and the efficiency of virion recovery following lysis. Dashed lines indicate score boundaries used to categorize variants as null-like, deleterious, wt-like, or hyper-infective.

Fig. 2. Comparison of empirical mutational tolerance and evolutionary diversity.

a Categorical effects of all amino acid substitutions. Categories are defined in Fig. 1e. The wt sequence of the J region is shown across the top and the amino acid substitutions on the Y axis. b Amino acid diversity, calculated from ConSurf [45], across 910 orthologs of J. Zero represents the mean diversity across positions. c For each amino acid position, the evolutionary diversity (y-axis) is compared to the average progeny produced by amino acid substitutions at that position (x-axis). Diversity of each position correlates only weakly with the average number of progeny (r² = 0.06, p < 0.01), in contrast to cellular proteins for which mutational tolerance and evolutionary diversity are more strongly related [24, 25, 28]. Gold points indicate positions where mutations have been reported that expand host range [10, 11]; these positions are more diverse but no more mutationally tolerant than the average position. Crosses represent 95% confidence intervals of the mean diversity and progeny of variants for positions with host range mutations (gold) or all positions (black). “Pos”: position.

Fig. 3. Selection of λ bearing J variants on λ-resistant hosts.

a Distribution of fitness effects on each host, expressed in terms of mean progeny produced per infectious cycle. In each case, the synonymous and nonsense distributions can be separated, despite the nominal λ-resistance of each lamB allele. Variants that outperform wild type λ do so by a larger margin on hosts that are more resistant. b Progeny for each J variant shown by the position of the mutation in the sequence. Adaptive mutations occur frequently at a subset of positions, but these positions are spread over the entire mutagenized region. Positions with many hyper-infective mutations are more apparent on the more resistant hosts LamB-R219H and LamB-T264I than on LamB-wt or LamB-G267D. c Correlation between progeny produced by λ bearing each J variant on its wild type host (y-axis), or on a host bearing a plasmid-borne lamB allele (x-axis). Variants that are highly infective on a non-wt host tend to also be infective on the wild type host, with some exceptions. However, many variants that are highly infective on the wild type host are poorly infective on non-wt hosts.

Fig. 4. Discrimination of promiscuity from specificity by comparison of variant effects across different hosts.

a Under a nested model, phages have more or less ability to generally infect hosts, which we describe as “promiscuity”, which contends with the level of resistance of potential hosts. b Under a lock-key model, the specific relationship between a host and phage determines infectivity, rather than host-independent properties of the phage or phage-independent properties of the host. c We can estimate the level of resistance of a given host by asking how well λ^wt produces progeny on it compared to LamB-wt (x-axis). Some J variants, like A84M are less affected by resistance than wild type λ, whereas others, like Q26S, are more affected. The black line represents synonymous variants, with error bars equal to ±1 standard deviation. We calculate promiscuity as the normalized Shannon’s entropy for each variant across the four potential hosts. d Some J variants confer infectivity on only a single host and are null-like on all others. Most of these variants, like Q96E, are deleterious, even on the host for which they are specific. Therefore, most of these variants cannot drive adaptation to a novel host by themselves, though they may work in concert with other variants. e Positions with promiscuous variants are shown in red with respect to their tolerance to mutation and amino acid diversity, with the size of the circle representing the number of unique promiscuous variants. The weighted average of these positions (cross, red) is more tolerant to mutation but not more diverse than the average of all positions (cross, black). Crosses represent 95% confidence intervals. f Variants that display specific infectivity on a single LamB variant fall at positions shown in orange. The weighted average of these positions has higher amino acid diversity, but is not more mutationally tolerant, than the average position.

Fig. 5. Double missense variants in J can mediate adaptation to new hosts.

a The double missense variant S30W, A94S is a combination of a promiscuous variant (A94S) and a LamB-G267D-specific variant (S30W). The double missense variant exhibits sign epistasis, improving infectivity on LamB-G267D despite the deleteriousness of each single missense variant. b For double missense variants (silver), we calculated the expected progeny from the progeny of each of the single missense variants using a simple multiplicative model. A subset of double missense variants strongly deviates from the multiplicative model, in contrast to variants in which a single missense mutation is paired with a single synonymous mutation (orange), which are more likely to agree with the multiplicative model (r² = 0.92 missense × synonymous vs. r² = 0.64 missense × missense). c Across the four hosts, most double missense variants in J do not exhibit significant epistasis (top left panel). However, double missense variants that contain a promiscuous variant, a host-specific variant, or both, are more likely to exhibit significant epistasis. We measured significant epistasis in 13.2% of double missense variants containing promiscuous variants, 5.4% containing host-specific variants, and 23.8% containing both, compared to 1.8% containing neither. d For each host, progeny is positively associated with promiscuity and with positive epistasis. However, these associations become more salient on resistant hosts, with all infective variants on LamB-R219H being promiscuous and positively epistatic, compared to a minority of variants on LamB-wt.