Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori

Structures and Dynamics of Native-State Transmembrane Protein Targets and Bound Lipids

Membrane proteins work within asymmetric bilayers of lipid molecules that are critical for their biological structures, dynamics and interactions. These properties are lost when detergents dislodge lipids, ligands and subunits, but are maintained in native nanodiscs formed using styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA) copolymers. These amphipathic polymers allow extraction of multicomponent complexes of post-translationally modified membrane-bound proteins directly from organ homogenates or membranes from diverse types of cells and organelles. Here, we review the structures and mechanisms of transmembrane targets and their interactions with lipids including phosphoinositides (PIs), as resolved using nanodisc systems and methods including cryo-electron microscopy (cryo-EM) and X-ray diffraction (XRD). We focus on therapeutic targets including several G protein-coupled receptors (GPCRs), as well as ion channels and transporters that are driving the development of next-generation native nanodiscs. The design of new synthetic polymers and complementary biophysical tools bodes well for the future of drug discovery and structural biology of native membrane:protein assemblies (memteins)

Mechanism of Tet(O)-mediated tetracycline resistance

Tet(O) is an elongation factor-like protein which confers resistance to the protein synthesis inhibitor tetracycline by promoting the release of the drug from its inhibitory site on the ribosome. Here we investigated the interaction of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS) probing and binding assays, that it interacts preferentially with the post-translocational ribosome. Furthermore, using an XTP-dependent mutant of Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within the ribosome which can be detected by EF-Tu, and manifested as a stimulation in the GTPase activity of this elongation factor. As such, these conformational changes probably involve the ribosomal GTPase-associated center and, accordingly, Tet(O) alters the DMS modification pattern of the L11 region. Additionally, tetracycline binding is associated with an E(a) of 58 kJ/mol. These results suggest a model where both Tet(O) and tetracycline induce a conformational change in functionally opposite directions and the Tet(O)-induced conformation persists after it has left the ribosome; this prevents rebinding of the drug while allowing productive A-site occupation by a ternary complex in the presence of tetracycline

The tetracycline resistance protein Tet() perturbs the conformation of the ribosomal decoding centre

Tet() is an elongation factor-like protein found in clinical isolates of Campylobacter jejuni that confers resistance to the protein-synthesis inhibitor tetracycline. Tet() interacts with the 70S ribosome and promotes the release of bound tetracycline, however, as shown here, it does not form the same functional interaction with the 30S subunit. Chemical probing demonstrates that Tet() changes the reactivity of the 16S rRNA to dimethyl sulphate (DMS). These changes cluster within the decoding site, where C1214 is protected and A1408 is enhanced to DMS reactivity. C1214 is close to, but does not overlap, the primary tetracycline-binding site, whereas A1408 is in a region distinct from the Tet() binding site visualized by cryo-EM, indicating that Tet() induces long-range rearrangements that may mediate tetracycline resistance. Tetracycline enhances C1054 to DMS modification but this enhancement is inhibited in the presence of Tet() unlike the tetracycline-dependent protection of A892 which is unaffected by Tet(). C1054 is part of the primary binding site of tetracycline and A892 is part of the secondary binding site. Therefore, the results for the first time demonstrate that the primary tetracycline binding site is correlated with tetracycline's inhibitory effect on protein synthesis