Metabolomics Analysis Identifies d-Alanine-d-Alanine Ligase as the Primary Lethal Target of d-Cycloserine in Mycobacteria

Abstract

D-cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR)- drug resistant strains of Mycobacterium tuberculosis. D-cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of Alanine racemase (Alr) and D-Alanine-D-alanine ligase (Ddl). Although, the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of D-alanyl-D-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of D-alanine thus allowing DCS to outcompete D-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce D-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.

Metabolomics: Applications and Promise in Mycobacterial Disease

“…Cycloserine inhibits several enzymes within the peptidoglycan biosynthesis pathways, particularly D-alanine-D-alanine ligase, a main enzyme of this pathway (63). These methods can be used to study the growth and replication of the organisms.…”

Multidimensional NMR approaches towards highly resolved, sensitive and high-throughput quantitative metabolomics

“…Absolute concentrations can then be calculated, provided that an internal standard of known concentration has been added to the sample. HSQC 0 has been successfully applied by Halouska et al to better understand the impact of D-cycloserine on mycobacteria [18]. This strategy is however limited to the HSQC sequence, and its overall sensitivity per unit of time is relatively limited since three spectra must be recorded in fully relaxed conditions for each sample.…”

Current therapies for the treatment of multidrug-resistant tuberculosis in children in India

“…It is bacteriostatic in action; main mechanism of action being inhibition of cell wall synthesis by competitive inhibition of alanine racemase and d-alanine-d-alanine ligase [37]. Neuropsychiatric side effects of cycloserine are mediated by NMDA receptors; they are usually dose and concentration dependent [38] A systematic review on treatment outcome of MDR-TB in children reported adverse events related to cycloserine in only 6 out of 182 children [39].…”