Evolutionary Trajectory of the Tet(X) Family: Critical Residue Changes towards High-Level Tigecycline Resistance

The emergence of the plasmid-mediated high-level tigecycline resistance mechanism Tet(X) threatens the role of tigecycline as the "last-resort" antibiotic in the treatment of infections caused by carbapenem-resistant Gram-negative bacteria. Compared with that of the prototypical Tet(X), the enzymatic activities of Tet(X3) and Tet(X4) were significantly enhanced, correlating with high-level tigecycline resistance, but the underlying mechanisms remain unclear. In this study, we probed the key amino acid changes leading to the enhancement of Tet(X) function and clarified the structural characteristics and evolutionary path of Tet(X) based upon the key residue changes. Through domain exchange and site-directed mutagenesis experiments, we successfully identified five candidate residues mutations (L282S, A339T, D340N, V350I, and K351E), involved in Tet(X2) activity enhancement. Importantly, these 5 residue changes were 100% conserved among all reported high-activity Tet(X) orthologs, Tet(X3) to Tet(X7), suggesting the important role of these residue changes in the molecular evolution of Tet(X). Structural analysis suggested that the mutant residues did not directly participate in the substrate and flavin adenine dinucleotide (FAD) recognition or binding, but indirectly altered the conformational dynamics of the enzyme through the interaction with adjacent residues. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and UV full-wavelength scanning experiments confirmed that each mutation led to an increase in activity without changing the biochemical properties of the Tet(X) enzyme. Further phylogenetic analysis suggested that Riemerella anatipestifer served as an important incubator and a main bridge vector for the resistance enhancement and spread of Tet(X). This study expands the knowledge of the structure and function of Tet(X) and provides insights into the evolutionary relationship between Tet(X) orthologs.IMPORTANCE The newly emerged tigecycline-inactivating enzymes Tet(X3) and Tet(X4), which are associated with high-level tigecycline resistance, demonstrated significantly higher activities in comparison to that of the prototypical Tet(X) enzyme, threatening the clinical efficacy of tigecycline as a last-resort antibiotic to treat multidrug-resistant (MDR) Gram-negative bacterial infections. However, the molecular mechanisms leading to high-level tigecycline resistance remain elusive. Here, we identified 5 key residue changes that lead to enhanced Tet(X) activity through domain swapping and site-directed mutagenesis. Instead of direct involvement with substrate binding or catalysis, these residue changes indirectly alter the conformational dynamics and allosterically affect enzyme activities. These findings further broaden the understanding of the structural characteristics and functional evolution of Tet(X) and provide a basis for the subsequent screening of specific inhibitors and the development of novel tetracycline antibiotics.