The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM with fewer mannan residues. To understand the importance of this protein, the antibiotic sensitivity of ∆lmeA was tested using a resazurin-based viability assay. We found that the lmeA deletion leads to increased sensitivities to antibiotics such as vancomycin and erythromycin, and lmeA overexpression leads to increased antibiotic resistance. To directly test if the increased antibiotic sensitivity is due to the defective permeability barrier, we used an ethidium bromide uptake assay and found that ∆lmeA is more efficient in taking up ethidium bromide in the cell. We have also found that LmeA is important for protein stabilization under stress conditions. MptA is an α1,6-mannosyltransferase involved in elongation of LM and LAM mannan chain. During stress conditions in the ΔlmeA mutant, levels of MptA decrease significantly relative to wild-type. This also results in delayed doubling time after stress, a phenotype not seen in this mutant under normal growth conditions. In addition, the ΔlmeA mutant has differential protein expression during stress conditions relative to ΔlmeA in log phase, or to wild-type in either condition. To help elucidate the role of LmeA at the molecular level, binding behavior of this protein to membrane fractions was determined. In a subcellular fractionation analysis, LmeA localizes to fractions containing plasma membrane, which is tightly bound to cell wall layers. To test the binding of LmeA to membrane further, LmeA was heterologously expressed in Escherichia coli, purified, and mixed M. smegmatis cell lysate. LmeA localized to intracellular domain fractions (IMD), indicating that LmeA is capable of localizing to fractions containing only plasma membrane. Consistent with this finding, LmeA is capable of binding to spheroplasts in both an ELISA setting as well as in a sucrose gradient fractionation setting. It has also been determined that ΔlmeA has a defective capsular layer with a unique phenotype relative to other strains. We have concluded that LmeA is important for antibiotic resistance, cell envelope permeability, capsule formation, stress response, and have also determined its binding properties.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-1980 |
| Date | 15 July 2020 |
| Creators | Osman, Sarah Hassan |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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