Studies into the role of peptidoglycan glycolylation in mycobacterial dormancy and resuscitation

A third of the global population are estimated to be latently infected with Mycobacterium tuberculosis (Mtb). This latent infection is most likely caused by non-replicating bacilli. Resuscitation-promoting factors (Rpfs) are secreted muralytic enzymes which are important for replication in vivo and are involved in resuscitation of dormant bacilli and reactivation of chronic tuberculosis. The precise mechanism of resuscitation remains unknown; however their enzymatic activity is essential for resuscitation and growth stimulatory effects. In mycobacterial peptidoglycan, muramic acid is present in acetylated and glycolylated forms. Glycolylation occurs in the cytoplasm during synthesis of peptidoglycan precursors by action of a UDP-N-acetylmuramic acid hydroxylase (NamH). The significance of glycolylation for mycobacterial growth and persistence is unknown. The overall aims of this study were to investigate the importance of peptidoglycan glycolylation in mycobacterial dormancy and resuscitation. Rpfs are the key enzymes involved in these processes, it was therefore predicted they might be adapted for recognition and cleavage of glycolylated peptidoglycan. Mtb NamH was over-expressed in E. coli in order to obtain glycolylated peptidoglycan and investigate its digestion by recombinant Rpfs. Recombinant Rpf was active by zymography but failed to release soluble muropeptides from different types of peptidoglycan. The role of peptidoglycan glycolylation in stimulation of mycobacterial growth and resuscitation of non-culturable bacilli was investigated in M. smegmatis: Mtb Rpfs were shown to stimulate growth in wild type and ΔnamH M. smegmatis, indicating that Rpf activity was not influenced by peptidoglycan glycolylation. Mtb Rpfs were also able to stimulate resuscitation of non-culturable M. smegmatis. The ΔnamH mutant failed to produce non-culturable cells, therefore its resuscitation could not be investigated. A ΔnamH Mtb mutant showed no significant difference in replication in vitro and in cultured macrophages; however it was more sensitive to isoniazid treatment. Overall these results indicate that peptidoglycan glycolylation is important for maintenance of cell wall structure and antimicrobial resistance.