New chemotherapeutic treatments are needed for Mycobacterium tuberculosis as it causes two million deaths annually and is emerging with drug-resistant strings. One focus of research is the micro bacterial cell wall, an effective barrier against antibiotics and host immune system. We previously reported that there are at least two membrane domains in mycobacteria, termed plasma membrane free of cell wall components (PMf) and plasma membrane with cell wall components (PM-CW).
These membrane domains are functionally distinct but are both rich in a phospholid called phosphatidylethanolamine (PE). MSMEG-4199 and MSMEG_6851, homologous to RV2140c and Rv1910c respectively in M. tuberculosis, have been annotated as PE-binding proteins in Mycobacterium smegmatis, a non-pathogenic model organism, indicating their potential role in lipid metabolism. As little is known about the sub cellular localization of these PE-binding proteins, we asked if these proteins are associated with any of these membrane fractions.
First, to examine the localization of the proteins in the cell wall, these genes were cloned and constructed into a plasmid vector to be expressed with specific epitope tags in M. smegmatis. Western blotting confirmed the expression of the expected proteins in lysates prepared by bead beating and nitrogen cavitation. Subcellular localization of the epitope tagged proteins was examined through sucrose gradient density fractionation. We found that MSMEG-4199 is enriched in the cytosol rather than PFf or PM-CW. Our data so far suggest that MSMEG-4199 may not function as a PE-binding protein. More detailed analysis is underway to determine potential roles of these proteins in lipid metabolism.