Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome

Abstract

Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.The tetracyclines are a group of broad-spectrum antibiotics which are generally considered to prevent bacterial growth by inhibiting protein synthesis. This results from binding of antibiotic to a single site in the 30S ribosomal subunit which prevents attachment of aminoacyl tRNA to the ribosomal acceptor site (3). In order to reach the ribosome, these antibiotics must traverse the hydrophobic lipid bilayer of the bacterial cytoplasmic membrane (3). At physiological pH tetracyclines can exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a high-energy, hydrophilic zwitterionic structure (7). A solvent-dependent equilibrium between the two forms has been demonstrated with oxytetracycline free base and is supported by X-ray analyses of tetracyclines crystallized from aqueous and nonaqueous solvents (7,15). Both forms are believed to be important for the antibacterial activity of tetracyclines, the low-energy, lipophilic conformational form (Fig. 1A) for uptake across the cytoplasmic membrane and the hydrophilic, zwitterionic structure (Fig. 1B) for binding to the ribosome (7).Chelocardin (14) is a naturally occurring anhydrotetracycline derivative with a modified A ring (Fig. 2). In contrast to the solvent-dependent equilibrium of the two tetracycline species mentioned above, chelocardin apparently exists in the same conformation in both polar and nonpolar solvents, as evidenced by circular dichroism measurements (6). We believe this is related to the planarity of the BCD rings in chelocardin and that a lipophilic form, perhaps related to that of tetracycline, is the preferred species. Thes...

tRNA modification activity is necessary for Tet(M)-mediated tetracycline resistance

“…None of the strains tested here exhibited resistance to chelocardin or anhydrotetracycline (data not shown), consistent with previous observations (9,32). The sensitivity of E. coli to anhydrotetracycline and chelocardin is not mediated at the level of protein synthesis (34).…”

Noninducible Tet repressor mutations map from the operator binding motif to the C terminus

“…In contrast to TetR DN95, the mutants TetR SG85 and CR144 are 88 and 92% inducible with anhydro-TC, but only 7 and 9% inducible by TC, respectively. This suggests different induction or binding mechanisms for TC and anhydro-TC which would resemble their different mechanisms of antibiotic activities reported recently (17). Only some of the TetRs mutations show a clearly reduced inducibility for anhydro-TC.…”

Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate

“…Tetracyclines were discovered in 1940s, and they have developed into a family of broad-spectrum antibiotics with activities against bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites ( 30 ). The bacteriostatic activity is caused by preventing the binding of aminoacyl tRNA to ribosomes, which is necessary for protein synthesis ( 31 ).…”