Date of Award
Master of Science (MS)
Microbiology and Immunology
Staphylococcus aureus is a major human pathogen known to cause disease in a wide range of tissues. In order to thrive in such diverse environments, S. aureus uses multiple adaptive traits such as trace metal/nutrient acquisition, shifts in metabolic activity, and expression of detoxification systems, all of which allow the bacterium to proliferate and survive in nutritionally deficient and inhospitable environments.
One essential metabolite used by S. aureus is lipoic acid, a cofactor of enzyme complexes used in aerobic metabolism, fatty acid biosynthesis, glycine detoxification, and maintenance of redox homeostasis. Prior studies in the lab used a genetic approach to define the lipoic acid biosynthesis and salvage pathways of S. aureus. These studies determined that S. aureus synthesizes lipoic acid from an octanoic acid precursor, or through salvage mechanisms, where lipoic acid is acquired from the environment by the action of lipoic acid ligases LplA1 and LplA2. In addition, it was demonstrated that LplA1, but not LplA2, is necessary for the salvage of lipoic acid in vitro, whereas both ligases are sufficient to promote infection of tissues in vivo. Because the LplA2 ligase does not have a discernable function in vitro, its exact role in lipoic acid salvage is unknown.
Based on this information, I hypothesized that LplA1 and LplA2 may stimulate growth by using alternate lipoylated substrates. To determine if the ligases use alternate sources of lipoic acid, I evaluated growth phenotypes by supplementing media with derivatives of lipoic acid. I
found that only LplA1, and not LplA2, can use free lipoic acid and peptide bound lipoic acid to stimulate bacterial growth in vitro. In order to further elucidate the functional differences and substrate usage of the ligases, I conducted lipoylation assays with purified recombinant ligases in the presence of lipoic acid, lipoamide, DKLA, and octanoic acid. My results indicated that LplA1 can directly use lipoic acid to lipoylate GcvH, GcvH-L and E2-OGDH, whereas LplA2 can directly use lipoic acid to lipoylate E2-PDH, E2-OGDH, E2-BCODH, as well as GcvH-L. These data suggest that both lipoic acid ligases in S. aureus have preferred targets for lipoylation and that they can act independently from one another.
Together, these studies highlight the importance of the divergent functions of LplA1 and LplA2 and may explain why S. aureus thrives so well when faced with low levels of free lipoic acid during host infection.
Laczkovich, Irina, "Defining the Roles of the Lipoic Acid Ligases in Promoting Staphylococcus Aureus Metabolic Homeostasis and Virulence" (2017). Master's Theses. 3688.
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Copyright © 2017 Irina Laczkovich
Available for download on Sunday, September 18, 2022