Investigating the role of GlfT2 in mycobacterial cell wall assembly

Abstract

The disease tuberculosis has remained difficult to treat to many small molecule treatments. One feature thought to contribute to this is the complex, impermeable cell wall of Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis. A core component of this cell wall is a linear carbohydrate polymer known as the galactan. The galactan, comprised of galactofuranose residues, serves as an essential covalent connector between the waxy mycolic acids bilayer and the peptidoglycan. In M. tb, the elongation of the galactan is mediated by a galactofuranosyl transferase, GlfT2. Using a combination of data derived from in vitro enzymatic assays and in vivo cell wall composition assays, we have arrived at the hypothesis that GlfT2 controls both length and linkage patterns of the galactan chain intrinsically. However, the hypothesis that GlfT2 is the only determining factor in length and linkage distribution has yet to be tested in vivo. The role of GlfT2 in galactan elongation is evolutionarily conserved; however, galactan length and linkage distribution varies across species. By transferring GlfT2 homologues from closely related organisms into Mycobacterium smegmatis (M. smeg) and studying the resulting growth patterns and cell walls, we can determine whether GlfT2 alone controls polymer length and linkages. To do this, we have developed and confirmed a GlfT2‐deficient strain of M. smeg using recombineering methods. We have also cloned a small library of GlfT2 homologues to be transformed into this strain. Preliminary results with Corynebacterium glutamicum (C. glut) GlfT2 have demonstrated rescued, but slower, growth in the deficient strain. We are particularly interested further assessment toward how changes to the galactan affect mycobacterial physiology.