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

• Published in: mSphere 2018-07, Vol.3 (4)
• Main Authors: Man, DeDe Kwun-Wai, Kanno, Tokuwa, Manzo, Giorgia, Robertson, Brian D, Lam, Jenny K W, Mason, A James
• Format: Article
• Language: English
• Subjects: Anisotropy; Anti-Infective Agents - metabolism; Antibiotics; Antimicrobial Cationic Peptides - metabolism; Antimicrobial peptides; Bacteria; Capreomycin; Capreomycin - metabolism; Cationic antimicrobial peptides; Cell Wall - metabolism; Discriminant analysis; Drug resistance; Drug Synergism; Fluorescence Polarization; Gram-negative bacteria; Investigations; Magnetic Resonance Spectroscopy; Membranes; Metabolites; Metabolomics; Mycobacterium smegmatis; Mycobacterium smegmatis - drug effects; Mycobacterium smegmatis - metabolism; Mycolic Acids - metabolism; NMR; Nuclear magnetic resonance; Oxidative stress; Peptides; Pharmaceutical sciences; Photoelectron Spectroscopy; Proline; Rifampin; Rifampin - metabolism; Software; Spectroscopy; Spectrum analysis; Surface properties; Therapeutics and Prevention; Trehalose; Tuberculosis; Volcanoes
• ISSN: 2379-5042; 2379-5042
• DOI: 10.1128/mSphere.00218-18

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 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 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 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. 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 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