Summary The burden of treating drug resistant TB and the emergence of new strains that are essentially resistant to all TB drugs has prompted the re-assessment of ways to treat M. tuberculosis disease. One such treatment modality is the use of the β-lactam class of antibiotics that target the peptide crosslinking pathways of bacterial peptidoglycan (PG). These antibiotics are not used to treat M. tuberculosis infections due to the inherent resistance of the bacteria via the BlaC β-lactamase. Nevertheless, M. tuberculosis can be killed by these antibiotics in combination with the β-lactamase inhibitor clavulanic acid. This observation, made by us and other groups, has renewed interest in exploiting β-lactam antibiotics as a way to treat TB infections. However, knowledge of PG biosynthesis in mycobacteria has lagged behind that of other bacteria. Our overall research goals are to learn more about the assembly and maintenance of mycobacterial peptidoglycan (PG). In this proposal, we are interested in the biological significance of novel 3-3 peptide crosslinks within the PG. These linkages, first described in M. tuberculosis decades ago, are found in many other bacteria but their significance in cell wall biology is poorly understood. The 3-3 crosslinks differ from classical 4-3 crosslinks, also found in mycobacteria, in that 3-3 crosslinks are catalyzed by a novel pathway that is distinct from the 4-3 crosslink pathway. While the 4-3 crosslinks are catalyzed by penicillin sensitive DD- transpeptidases (also called penicillin-binding proteins or PBPs), the 3-3 crosslinks are made by unique, penicillin insensitive LD-transpeptidases (Ldts). Our previous work has demonstrated that the mycobacterial Ldt enzymes can be grouped into specific classes based upon sequence identity. Using M. smegmatis as a surrogate organism we have constructed mutants with either single or multiple deletions of each of the ldt genes and showed that only certain combinations of mutations yield mutant phenotypes. Central to all mutants with a substantial phenotype is loss of the Class 5 ldtC gene, suggesting that LdtC is a key enzyme in this pathway. This application aims to further examine the role of Ldt enzymes in M. tuberculosis cell wall biology, with a particular focus on LdtC, and to characterize the role of DD-carboxypeptidases in the 3-3 crosslink pathway.