Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes

Berglund, Fanny; Böhm, Maria Elisabeth; Martinsson, Anton; Ebmeyer, Stefan; Österlund, Tobias; Johnning, Anna; Larsson, D. G. Joakim; Kristiansson, Erik

doi:10.1099/mgen.0.000455

Cited by 27 publications

(27 citation statements)

References 76 publications

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“…Genes encoding tetracycline-degrading enzymes and their homologues are widely present in the natural environment and in human and animal gut metagenomes ( 30 , 31 ). Tetracycline-degrading enzymes, including Tet(X), are considered to be evolutionarily originated from the environment, while mobile genetic elements promote their transfer and spread into different biological niches ( 2 ).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…It has been shown, for example, that knocking out the tigecycline-resistant genes in R. anatipestifer results in a decrease in bacterial metabolic activity [ 68 ]. In general, however, the proportion of the tet (X) genes is quite small within the known and computed diversity of all tetracycline-resistant genes [ 69 ]. Additionally, there is no indication of tet (X) entry into Gram-positive bacteria.…”

Section: Emergence and Rise Of Amr: From Chromosomal Mutations To Acquired Mobile Resistancementioning

confidence: 99%

Acquisition and Spread of Antimicrobial Resistance: A tet(X) Case Study

Aminov

2021

IJMS

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Understanding the mechanisms leading to the rise and dissemination of antimicrobial resistance (AMR) is crucially important for the preservation of power of antimicrobials and controlling infectious diseases. Measures to monitor and detect AMR, however, have been significantly delayed and introduced much later after the beginning of industrial production and consumption of antimicrobials. However, monitoring and detection of AMR is largely focused on bacterial pathogens, thus missing multiple key events which take place before the emergence and spread of AMR among the pathogens. In this regard, careful analysis of AMR development towards recently introduced antimicrobials may serve as a valuable example for the better understanding of mechanisms driving AMR evolution. Here, the example of evolution of tet(X), which confers resistance to the next-generation tetracyclines, is summarised and discussed. Initial mechanisms of resistance to these antimicrobials among pathogens were mostly via chromosomal mutations leading to the overexpression of efflux pumps. High-level resistance was achieved only after the acquisition of flavin-dependent monooxygenase-encoding genes from the environmental microbiota. These genes confer resistance to all tetracyclines, including the next-generation tetracyclines, and thus were termed tet(X). ISCR2 and IS26, as well as a variety of conjugative and mobilizable plasmids of different incompatibility groups, played an essential role in the acquisition of tet(X) genes from natural reservoirs and in further dissemination among bacterial commensals and pathogens. This process, which took place within the last decade, demonstrates how rapidly AMR evolution may progress, taking away some drugs of last resort from our arsenal.

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“… 2–5 A recent in silico analysis of genomic and metagenomic databases identified 116 distinct families of putative RPP genes. 6 Six of these were expressed from a plasmid vector in Escherichia coli , of which one gave a strong tetracycline resistance phenotype, two had a marginal effect on resistance and three gave no resistance. 6 The mechanism of resistance has been characterized for two RPP proteins, Tet(M) and Tet(O).…”

Section: Introductionmentioning

confidence: 99%

“… 6 Six of these were expressed from a plasmid vector in Escherichia coli , of which one gave a strong tetracycline resistance phenotype, two had a marginal effect on resistance and three gave no resistance. 6 The mechanism of resistance has been characterized for two RPP proteins, Tet(M) and Tet(O). Each protects the ribosome by displacing tetracycline from the A site, thereby allowing the ternary complex to bind and protein synthesis to continue.…”

Section: Introductionmentioning

confidence: 99%

Phenotypic and genetic barriers to establishment of horizontally transferred genes encoding ribosomal protection proteins

Yadav

Garoff

Huseby

et al. 2021

Journal of Antimicrobial Chemotherapy

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Background Ribosomal protection proteins (RPPs) interact with bacterial ribosomes to prevent inhibition of protein synthesis by tetracycline. RPP genes have evolved from a common ancestor into at least 12 distinct classes and spread by horizontal genetic transfer into a wide range of bacteria. Many bacterial genera host RPP genes from multiple classes but tet(M) is the predominant RPP gene found in Escherichia coli. Objectives We asked whether phenotypic barriers (low-level resistance, high fitness cost) might constrain the fixation of other RPP genes in E. coli. Methods We expressed a diverse set of six different RPP genes in E. coli, including tet(M), and quantified tetracycline susceptibility and growth phenotypes as a function of expression level, and evolvability to overcome identified phenotypic barriers. Results The genes tet(M) and tet(Q) conferred high-level tetracycline resistance without reducing fitness; tet(O) and tet(W) conferred high-level resistance but significantly reduced growth fitness; tetB(P) conferred low-level resistance and while mutants conferring high-level resistance were selectable these had reduced growth fitness; otr(A) did not confer resistance and resistant mutants could not be selected. Evolution experiments suggested that codon usage patterns in tet(O) and tet(W), and transcriptional silencing associated with nucleotide composition in tetB(P), accounted for the observed phenotypic barriers. Conclusions With the exception of tet(Q), the data reveal significant phenotypic and genetic barriers to the fixation of additional RPP genes in E. coli.

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Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes

Cited by 27 publications

References 76 publications

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

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

Acquisition and Spread of Antimicrobial Resistance: A tet(X) Case Study

Phenotypic and genetic barriers to establishment of horizontally transferred genes encoding ribosomal protection proteins

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