Functional and phylogenetic analysis of TetX variants to design a new classification system

Abstract

Recently, many TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. In this study, we identified essential residues including 329, 339, 340, 350, and 351 in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into TetX-A class, TetX-B class and TetX-C class. We further found that TetX-A class variants originated from Bacteroidetes, with some variants further evolving to TetX-C class and acquired by Enterobacteriaceae. On the other hand, our data showed that some variants genes belonging to TetX-A class evolved directly to TetX-B class, which was further transmitted to Acinetobacter spp. This new classification system may facilitate better clinical management of patients infected by TetX-producing strains.

Fig. 1. Alignment of the complete amino acid sequences encoded by Tet(X) variants genes using BioEdit.

Amino acid residues are depicted in different color, the same amino acid is shown as dots in the alignment. Commonly mutated sites in Tet(X3), Tet(X4), Tet(X5), Tet(X6), and Tet(X7) compared with original TetX protein are highlighted in red box.

Fig. 2. Location of related residues in the Tet(X)-tigecycline complex structure.

a Mapping the test amino acid substitution sites in the FAD-binding domain (deep olive), substrate-binding domain (cyan), and C-terminal helix (red). Residues are depicted as pink stick. b FAD and substrate-binding sites and mutation residues are showed in the model of Tet(X2) mutant (V³²⁹M/A³³⁹T/D³⁴⁰N/V³⁵⁰I/K³⁵¹E). Loop 5 and mutant residues are shown as deep blue. Substrate-binding sites are depicted as green stick.

Fig. 3. Phylogenetic analysis of TetX like protein.

a TetX-related proteins are divided into three classes: TetX-A (green), TetX-B (yellow), and TetX-C (orange). Phylogeny is inferred by using the maximum-likelihood method and Flu +G + I model. A discrete Gamma distribution approach was used to depict the difference in evolutionary rate among the sites [4 categories (Gamma shape parameter = 0.609)]. This analysis involved 128 amino acid sequences. Different species hits the protein sequences organized in the phylum level. Bacteroidetes, Proteobacteria, Firmicutes, Spirochetes, Bacteroidetes plus Proteobacteria, and unclassified organisms are shown in blue, pink, light purple, and gray, respectively. Evolutionary analyses were conducted in online PlyML 3.0. The tree was visualized using iTOL (ITEREACTIVE TREE OF LIFE). b Phylogenetic analysis of the amino acid sequences of the reported TetX variants. The maximum-likelihood tree was inferred using MEGA X. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. c Reported TetX variants are distributed in TetX-A class, TetX-B class, and TetX-C class with specific substitutions.