Peptidoglycan Synthesis and Rod Shape Maintenance in Mycobacteria

Abstract

Bacteria surround themselves with a cell wall whose foundation is a layer called peptidoglycan. Peptidoglycan is a mesh of linear glycan strands that are linked together by short peptide side chains. As a crucial bacterial polymer, peptidoglycan is the target of numerous antibiotics. My thesis has focused on features of peptidoglycan in mycobacteria, a genus including the formidable human pathogen Mycobacterium tuberculosis. In comparison to other well-studied rod-shaped bacteria like Escherichia coli and Bacillus subtilis, mycobacteria are unique. First, the peptidoglycan of mycobacteria is highly enriched for specific linkages called 3-3 crosslinks. Second, mycobacteria grow via insertion of new peptidoglycan at the poles of the bacillus. This occurs at unequal rates depending on the age of the pole. While the lateral mode of growth in E. coli and B. subtilis ensures that new and old cell wall are constantly intermingled, mycobacterial polar growth segregates peptidoglycan by age whereby the newest material is at the poles and the oldest material is located toward mid-cell. L,D-transpeptidases are peptidoglycan synthesis enzymes that catalyze 3-3 crosslinks. As these crosslinks are rare in the model bacterial species from which we have garnered much of our knowledge about peptidoglycan, the role of this crosslink is not well understood. I discovered that 3-3 crosslinks are required to maintain rod shape at sites of aging cell wall in Mycobacterium smegmatis. Moreover, I found that uneven polar growth, and the subsequent spatial segregation of aging peptidoglycan, leads to an asymmetric distribution of peptidoglycan chemistries and enzymes, like penicillin binding proteins and L,D-transpeptidases, within a single cell. Lastly, I demonstrated that in the absence of L,D-transpeptidases, mycobacterial cells rely more heavily on penicillin binding proteins, peptidoglycan synthases that catalyze 4-3 crosslinks. Current first line therapies for tuberculosis target the mycobacterial cell wall, however they do not yet target the peptidoglycan layer. Non-carbapenem -lactams (with -lactamase inhibitors) and carbapenems, drugs that inhibit peptidoglycan synthases like penicillin binding proteins and L,D-transpeptidases, have garnered recent interest for the treatment of tuberculosis. My work on the spatial and genetic relationship between penicillin binding proteins and L,D-transpeptidases suggests details on the mechanism by which the combination of these antibiotics may kill tuberculosis faster than these drugs do alone. As resistance to current tuberculosis therapies continues to be a problem, it is critical that we gather mechanistic insights into the action of therapies to both aid in the rational combination of drugs, and to prepare ourselves for the putative mechanisms of resistance that will likely arise.