Rifampicin or capreomycin induced remodelling of theMycobacterium smegmatismycolic acid layer is mitigated in synergistic combinations with cationic antimicrobial peptides

Abstract

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 rifampicin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs; 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 (HR-MAS) NMR of bacteria, diphenylhexatriene (DPH) fluorescence anisotropy and laurdan emission spectroscopy we show that M. smegmatis responds to challenge with rifampicin or capreomycin by substantially altering its metabolism and, in particular, by remodelling the cell envelope. In NMR spectra of bacteria, reductions in intensity for mycolic acid lipid -(CH2)-, -CH3, R2CH-COOH, R2CH-OH and also -CH2-(CH=CH)-and -CH=CH-resonances were observed following challenge with rifampicin and capreomycin, while the latter also caused an increase in trehalose. These changes are consistent with a reduction of trehalose dimycolate and increase of trehalose monomycolate and are associated with an increase in rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampicin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes respectively in these metabolites and did not induce a significant increase in rigidity of the mycolic acid layer. Further, the response to rifampicin or capreomycin was significantly reduced when these were combined respectively with D-LAK120-HP13 and D-LAK120-A, suggesting a possible mechanism for the synergy of these combinations. The remodelling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampicin or capreomycin, but this can be mitigated, and rifampicin or capreomycin efficacy potentiated, by combining with AMPs.