Catharine A. Trieber

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 microg/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 microg/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.

The Molecular Basis for Cyclopiazonic Acid Inhibition of the Sarcoplasmic Reticulum Calcium Pump

The sarcoplasmic reticulum Ca2+-ATPase is essential for calcium reuptake in the muscle contraction-relaxation cycle. Here we present structures of a calcium-free state with bound cyclopiazonic acid (CPA) and magnesium fluoride at 2.65Å resolution and a calcium-free state with bound CPA and ADP at 3.4Å resolution. In both structures, CPA occupies the calcium access channel delimited by transmembrane segments M1–M4. Inhibition of Ca2+-ATPase is stabilized by a polar pocket that surrounds the tetramic acid of CPA and a hydrophobic platform that cradles the inhibitor. The calcium pump residues involved include Gln56, Leu61, Val62, and Asn101. We conclude that CPA inhibits the calcium pump by blocking the calcium access channel and immobilizing a subset of transmembrane helices. In the E2(CPA) structure, ADP is bound in a distinct orientation within the nucleotide binding pocket. The adenine ring is sandwiched between Arg489 of the nucleotide-binding domain and Arg678 of the phosphorylation domain. This mode of binding conforms to an adenine recognition motif commonly found in ATP-dependent proteins.

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 Ea 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.

Localization of the Ribosomal Protection Protein Tet(O) on the Ribosome and the Mechanism of Tetracycline Resistance

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.

Ribosomal Protection from Tetracycline Mediated by Tet(O): Tet(O) Interaction with Ribosomes Is GTP-Dependent

Tet(O) mediates tetracycline resistance by protecting the ribosome from inhibition. A recombinant Tet(O) protein with a histidine tag was purified and its activity in protein synthesis characterized. Tetracycline inhibited the rate of poly(Phe) synthesis, producing short peptide chains. Tet(O)-His was able to restore the elongation rate and processivity. 70S ribosomes bound tetracycline with high affinity. Tet(O)-His in the presence of GTP, but not GDP or GMP, reduced the affinity of the ribosomes for tetracycline. Non-hydrolyzable GTP analogs in the presence of the factor were also able to interfere with tetracycline binding. Ribosomes increased the affinity of Tet(O)-His for GTPgammaS. Tet(O), 70S ribosomes and GTPgammaS formed a complex that could be isolated by gel filtration. The GTP conformer is the active form of Tet(O) that interacts with the ribosome. GTP binding is necessary for Tet(O) activity.

Mechanism of Action of the Novel Aminomethylcycline Antibiotic Omadacycline

ABSTRACT Omadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus (MRSA), β-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae, Haemophilus influenzae, and Legionella. In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitro in the presence of the ribosomal protection protein Tet(O).

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 tetracyclines inhibitory effect on protein synthesis.

Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A

Background: Heterozygous mutations in the cytoplasmic domain of phospholamban cause lethal dilated cardiomyopathy. Results: The mutations alter phospholamban-protein kinase A interactions that are essential for substrate recognition and phosphorylation. Conclusion: Hereditary mutations in phospholamban that prevent phosphorylation by protein kinase A will lead to chronic inhibition of SERCA. Significance: Arginines in the cytoplasmic domain of phospholamban should be considered hot spots for hereditary mutations leading to dilated cardiomyopathy. The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg9 to Cys, Arg9 to Leu, Arg9 to His, and Arg14 deletion, alter regulation of SERCA. Deletion of Arg14 disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg9 causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg9 in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

Effects of phospholamban transmembrane mutants on the calcium affinity, maximal activity, and cooperativity of the sarcoplasmic reticulum calcium pump.

Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.

Consequences of Removal of a Molybdenum Ligand (DmsA-Ser-176) of Escherichia coli Dimethyl Sulfoxide Reductase

We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli dimethyl sulfoxide (Me2SO) reductase (DmsABC). Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me2SO and in vitro benzyl viologen:Me2SO oxidoreductase activity. EPR spectroscopy reveals changes in the line shape and the gav of the Mo(V) signals of the S176A and S176C enzymes. The midpoint potentials (Em,7) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are −15 and −175 mV. The Em,7 of the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide. Titration of the S176C mutant produced several overlapping Mo(V) species occurring at Eh > −150 mV, suggesting heterogeneity in the molybdenum environment. A Mo(V) spectrum was not visible in S176H membranes poised between −435 to 350 mV or oxidized with 200 µM ferricyanide. No differences were detected in the EPR spectra of the reduced [4Fe-4S] clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced [4Fe-4S] clusters.