Use of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria: role of D-alanine racemase

Abstract

D-Cycloserine (DCS) is only used with multidrug-resistant strains of tuberculosis because of serious side effects. DCS is known to inhibit cell wall biosynthesis, but the in vivo lethal target is still unknown. We have applied NMR-based metabolomics combined with principal component analysis to monitor the in vivo effect of DCS on Mycobacterium smegmatis. Our analysis suggests DCS functions by inhibiting multiple protein targets.

Figure 1

Alanine racemase converts L-alanine to D-alanine in the alanine pathway. D-alanyl-D-alanine ligase binds two molecules of D-alanine by peptide bonds, which is then used in the peptidoglycan biosynthesis pathway to form the cell wall.

Figure 2

Wild type mc²155 (A-C) and mutants GPM 14 (D-F), GPM 16 (G-I), TAM 23 (J-L), and TAM23 pTAMU3 (M-O) cells were plated on MADC (A,D,G,J), MADC with 50 mM D-alanine (B,E,H,K), MADC with 50 μg/ml DCS (C,F,I), or MADC with 1.73 μg/ml DCS (L). Plates were incubated at 37°C until colony diameters were c.a. 1.0 cm and colony images were digitally captured, cropped, and scaled to similar sizes. Each picture has a scale bar (5.0 mm) to indicate the actual colony size.

Figure 3

1D ¹H NMR spectra in order from bottom to top are: TAM23, mc²155, TAM23 with DCS, and mc²155 with DCS.

Figure 4

PCA scores plot comparing mc²155 ( ), TAM23 (●), GPM14 ( ), GPM16 ( ),TAM23 pTAMU3 ( ) mc²155 with DCS ( ), and TAM23 with DCS ( ), GPM14 with DCS ( ), GPM16 with DCS ( ), and TAM23 pTAMU3 with DCS ( ).