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. 2011 Sep;7(9):e1002184.
doi: 10.1371/journal.pcbi.1002184. Epub 2011 Sep 22.

Network Models of TEM β-Lactamase Mutations Coevolving Under Antibiotic Selection Show Modular Structure and Anticipate Evolutionary Trajectories

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Free PMC article

Network Models of TEM β-Lactamase Mutations Coevolving Under Antibiotic Selection Show Modular Structure and Anticipate Evolutionary Trajectories

Violeta Beleva Guthrie et al. PLoS Comput Biol. .
Free PMC article

Abstract

Understanding how novel functions evolve (genetic adaptation) is a critical goal of evolutionary biology. Among asexual organisms, genetic adaptation involves multiple mutations that frequently interact in a non-linear fashion (epistasis). Non-linear interactions pose a formidable challenge for the computational prediction of mutation effects. Here we use the recent evolution of β-lactamase under antibiotic selection as a model for genetic adaptation. We build a network of coevolving residues (possible functional interactions), in which nodes are mutant residue positions and links represent two positions found mutated together in the same sequence. Most often these pairs occur in the setting of more complex mutants. Focusing on extended-spectrum resistant sequences, we use network-theoretical tools to identify triple mutant trajectories of likely special significance for adaptation. We extrapolate evolutionary paths (n = 3) that increase resistance and that are longer than the units used to build the network (n = 2). These paths consist of a limited number of residue positions and are enriched for known triple mutant combinations that increase cefotaxime resistance. We find that the pairs of residues used to build the network frequently decrease resistance compared to their corresponding singlets. This is a surprising result, given that their coevolution suggests a selective advantage. Thus, β-lactamase adaptation is highly epistatic. Our method can identify triplets that increase resistance despite the underlying rugged fitness landscape and has the unique ability to make predictions by placing each mutant residue position in its functional context. Our approach requires only sequence information, sufficient genetic diversity, and discrete selective pressures. Thus, it can be used to analyze recent evolutionary events, where coevolution analysis methods that use phylogeny or statistical coupling are not possible. Improving our ability to assess evolutionary trajectories will help predict the evolution of clinically relevant genes and aid in protein design.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The TEM coevolution network and its communities.
The network was constructed based on frequencies of co-occurring mutated residue positions in 363 mutant TEM β-lactamase sequences. Node size is proportional to how well connected a node is to its neighbors and how many neighbors it has (weighted degree centrality, Methods). Link thickness is proportional to the number of sequences in our database in which both positions are mutated, normalized by the number of sequences in which only one or the other position is mutated (Methods). Node (residue) numbers are shown in Ambler notation. The Clauset community-finding algorithm identified three major communities, corresponding to three Bush-Jacobi β-lactamase phenotype classes: broad-spectrum antibiotic resistance or 2b (gray), extended-spectrum antibiotic resistance or 2be (blue) and inhibitor resistance or 2br (orange). Mutated positions with phenotypic effects documented in : extended-spectrum resistance 51, 173, 237, 240, 39, 164, 104, 238, 153, 265, 92, 224; inhibitor resistance 165, 69, 275, 276, 244, 201; inhibitor and extended-spectrum resistance: 182, 268. Image created with CytoScape .
Figure 2
Figure 2. The TEM extended-spectrum community network and its two subcommunities.
The network was constructed in the same way as Figure 1, but here we only used sequences associated with extended-spectrum antibiotic resistance. We identified two large subcommunities, the first containing the active-site residue 238 (light-blue), and the second containing the active-site residue 164 (dark-blue). Node size is proportional to how well connected a node is to its neighbors and how many neighbors it has (weighted degree centrality, Methods). Link thickness indicates how frequently two residues (nodes) are mutated in the same sequence, normalized by the number of sequences in which only one or the other position is mutated (Methods). Image created with CytoScape .
Figure 3
Figure 3. Cefotaxime plate growth assays for selected clones.
Cultures of cells expressing the β-lactamase mutants listed at the top of the gradients were stamped on LB plates containing a cefotaxime gradient. The direction of the gradient is from top (minimal concentration) to bottom (maximal concentration). The maximal concentration of the gradient is listed at the bottom. Note that in part B more than one concentration is shown to cover the wide range of resistance phenotypes of the panel of mutants being tested. (A) Two mutant triplets predicted to be of special significance by our analysis but that were not present in the sequence database used to build the network but were subsequently reported in , and a third triplet also predicted by our analysis but that showed only a marginal increase. Only the doublet with the highest level of resistance is shown. (B) Triplets with the strongest negative epistatic effects. The mutation responsible for the negative effect is highlighted in bold.
Figure 4
Figure 4. Pairwise epistatic interactions in the TEM extended-spectrum community either previously described in the literature or identified in our experiments , –.
Network is represented as in Figure 2. The subcommunity containing the active-site residue 238 is light blue and the subcommunity containing the active-site residue 164 is dark blue. Node size is proportional to weighted degree centrality (Methods). Link thickness indicates how frequently two residues (nodes) are mutated in the same sequence, normalized by the number of sequences in which only one or the other position is mutated (Methods). (A) Black links indicate positive epistatic interactions. (B) Red links indicate negative epistatic interactions. Because the network is constructed from co-occurring mutated residue pairs, negative-epistatic pairs may be underrepresented in or absent from the network, e.g. 39 and 173. Image created with CytoScape .

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