Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

Binding energies of the drugs capreomycin and streptomycin in complex with tuberculosis bacterial ribosome subunits.

Despite advances, tuberculosis remains a significant infectious disease, whose mortality presents alarming numbers. Although it can be cured, the number of cases of antimicrobial resistant strains is increasing, requiring the use of less efficient second-line drugs. Capreomycin and streptomycin are part of this group, being antibiotics whose mechanism of action is the inhibition of protein synthesis when interacting with the tuberculosis bacterial ribosome. Their binding mechanisms are distinct: capreomycin is able to bind to both ribosomal (30S and 50S) subunits, whereas streptomycin binds only to the smaller one (30S). In this context, the biochemical characterization of these binding sites for a proper understanding of their complex interactions is of crucial importance to increase their efficacy. Through crystallographic data and computer simulations, in this work we calculated the interaction binding energies of capreomycin and streptomycin in complex with the tuberculosis bacterial ribosome subunits, by using density functional theory (DFT) within the molecular fractionation with conjugated caps (MFCC) approach. For capreomycin in the 30S (50S) subunit, we investigated the binding energies of 44 (30) residues presented within a pocket radius of 14 Å (30 Å). Regarding streptomycin, 60 nucleotide (25 amino acid) residues distributed up to 12.5 Å (15 Å) away from the drug in the 30S subunit (S12 protein) were taken into account. We also identify the contributions of hydrogen bonds and hydrophobic interactions in the drug-receptor complex, and the regions of the drugs that most contributed to the anchorages of them in their binding sites, as well as identify residues that are most associated with mutations.

Rifampin- or Capreomycin-Induced Remodeling of the Mycobacterium smegmatis Mycolic Acid Layer Is Mitigated in Synergistic Combinations with Cationic Antimicrobial Peptides.

The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here, we investigate the response of Mycobacterium smegmatis to the presence of rifampin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs that are distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) metabolomics, diphenylhexatriene (DPH) fluorescence anisotropy measurements, and laurdan emission spectroscopy, we show that M. smegmatis responds to challenge with rifampin or capreomycin by substantially altering its metabolism and, in particular, by remodeling the cell envelope. Overall, the changes are consistent with a reduction of trehalose dimycolate and an increase of trehalose monomycolate and are associated with increased rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes, respectively, in mycomembrane metabolites and did not induce a significant increase in the rigidity of the mycolic acid layer. Furthermore, the response to rifampin or capreomycin was significantly reduced when these were combined with D-LAK120-HP13 and D-LAK120-A, respectively, suggesting a possible mechanism for the synergy of these combinations. The remodeling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampin or capreomycin, but this can be mitigated and the efficacy of rifampin or capreomycin potentiated by combining the drug with AMPs.IMPORTANCE We have used a combined NMR metabolomics/biophysical approach to better understand differences in the mechanisms of two closely related antimicrobial peptides, as well as the response of the model organism Mycobacterium smegmatis to challenge with first- or second-line antibiotics used against mycobacterial pathogens. We show that, in addition to membrane damage, the triggering of oxidative stress may be an important part of the mechanism of action of one AMP. The metabolic shift that accompanied rifampin and, particularly, capreomycin challenge was associated with modest and more dramatic changes, respectively, in the mycomembrane, providing a rationale for how the response to one antibiotic may affect bacterial penetration and, hence, the action of another. This study presents the first insights into how antimicrobial peptides may operate synergistically with existing antibiotics whose efficacy is waning or sensitize MDR mycobacteria and/or latent mycobacterial infections to them, prolonging the useful life of these drugs.

Mycobacterium smegmatis MSMEG_3705 encodes a selective major facilitator superfamily efflux pump with multiple roles.

Mycobacterium smegmatis mc(2)155 MSMEG_3705 gene was annotated to encode a transporter protein that contains 12 alpha-helical transmembrane domains. We predicted MSMEG_3705 encoding a major facilitator superfamily (MFS) member. To confirm the prediction, the M. smegmatis mc(2)155 MSMEG_3705 gene was deleted. The MSMEG_3705 deletion mutant strain M. smegmatis mc(2)155 ∆MSMEG_3705 was more sensitive to capreomycin. Moreover, M. smegmatis mc(2)155 ∆MSMEG_3705 strain accumulated more ethidium bromide intracellular than wild-type M. smegmatis mc(2)155. Quite unexpectedly, M. smegmatis mc(2)155 ∆MSMEG_3705 grew faster than the wild-type M. smegmatis mc(2)155. The upregulation of the expression of MSMEG_3706, a gene encoding isocitrate lyase downstream MSMEG_3705, in the deletion mutant, might underlie such faster growth in the mutant. The study showed that MSMEG_3705 encodes a genuine MFS member and plays significant role in bacterial growth and antibiotics resistance.

Unexpected N-acetylation of capreomycin by mycobacterial Eis enzymes.

OBJECTIVES: The enhanced intracellular survival (Eis) protein from Mycobacterium tuberculosis (Eis_Mtb), a regio-versatile N-acetyltransferase active towards many aminoglycosides (AGs), confers resistance to kanamycin A in some cases of extensively drug-resistant tuberculosis (XDR-TB). We assessed the activity of Eis_Mtb and of its homologue from Mycobacterium smegmatis (Eis_Msm) against a panel of anti-tuberculosis (TB) drugs and lysine-containing compounds. METHODS AND RESULTS: Both enzymes acetylated capreomycin and some lysine-containing compounds, but not other non-AG non-lysine-containing drugs tested. Modelling studies predicted the site of modification on capreomycin to be one of the two primary amines in its ß-lysine side chain. Using Eis_Mtb, we established via nuclear magnetic resonance (NMR) spectroscopy that acetylation of capreomycin occurs on the ε-amine of the ß-lysine side chain. Using Msm, we also demonstrated for the first time to our knowledge that acetylation of capreomycin results in deactivation of the drug. CONCLUSIONS: Eis is a unique acetyltransferase capable of inactivating the anti-TB drug capreomycin, AGs and other lysine-containing compounds.

Capreomycin--a polypeptide antitubercular antibiotic with unusual binding properties toward copper(II).

Capreomycin is an important therapeutic agent having intriguing and diverse molecular features. Its polypeptidic structure rich in nitrogen donors makes the drug a promising chelating agent for a number of transition metal ions, especially for copper(II). The results of the model investigational studies suggest that capreomycin anchors Cu(2+) ion with an amino function of the α,ß-diaminopropionic acid residue at pH around 5. At physiological pH copper(II) ion is coordinated by two deprotonated amide nitrogen atoms of the α,ß-diaminopropionic acid, the serine residue as well as the amino function deriving from the ß-lysine. Above that pH value we observe a rearrangement within the coordination sphere leading to movement of Cu(2+) to the center of the peptide ring with concurrent coordination of four nitrogen donors. Spin-lattice relaxation enhancements and potentiometric measurements clearly indicate that deprotonated amide nitrogen atom from the ß-ureidodehydroalanine moiety is the fourth donor atom.

Molecular basis for the selectivity of antituberculosis compounds capreomycin and viomycin.

Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.

The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome.

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-A resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.