Identification and Elucidation of the Roles of Lipoteichoic Acid Tailoring Enzymes in the Cell Envelope of Staphylococcus aureus and Other Firmicutes
Open Access
- Author:
- Kho, Kelvin
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 14, 2020
- Committee Members:
- Timothy Charles Meredith, Dissertation Advisor/Co-Advisor
Timothy Charles Meredith, Committee Chair/Co-Chair
Ken Keiler, Committee Member
B Tracy Nixon, Committee Member
Scott A Showalter, Outside Member
Timothy Iwao Miyashiro, Committee Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- Staphylococcus aureus
Firmicutes
Cell envelope
Teichoic acid
Lipoteichoic acid
Glycosylation
Gram-positive - Abstract:
- Staphylococcus aureus, like all Gram-positive bacteria, is characterized by a thick cell envelope. This envelope is comprised mainly of peptidoglycan and teichoic acids. Although peptidoglycan is the main structural component, teichoic acids are responsible for regulating various cell envelope processes including cell wall remodeling, protein secretion and nutrient sequestration. Moreover, these cell wall polymers are generally important for pathogenicity but are largely dispensable in vivo. Many antibiotics in clinical use target peptidoglycan biosynthetic pathway but there is growing interest in identifying inhibitors of teichoic acid biosynthesis as potentiators of pre-existing antibiotics. Recent approaches include high-throughput screening of large chemical compound libraries as well as the use of transposon insertion sequencing. These studies have yielded insights into the genes and phenotypes associated with teichoic acids as well as numerous synthetic lethal interactions. Among these are the conditional essentiality of SAOUHSC_00618 in the absence of tunicamycin. Here, we further identified other cellular roles of SAOUHSC_00618 including its roles as an effector of the staphylococcal accessory gene regulator system. Lipoteichoic acids (LTA) are anchored to the cell membrane by a diglucosyl-diacylglycerol moiety whereby their repeat units can vary from simply polyglycerol phosphates to more complex ones containing multiple saccharides and sidechains. Staphylococcus aureus expresses the former and like many other bacteria, decorates its teichoic acids with D-alanine esters and glycosyls. These different modifications are responsible for modulating many of the functions associated with teichoic acids. The different enzymes in teichoic acid biosynthesis including D-alanylation has been identified along with the multifaceted roles of teichoic acid D-alanylation. The LTA glycosylation pathway, however, remained unknown. Other Gram-positive bacteria such as Bacillus subtilis exhibit high levels of LTA glycosylation but the glycosylation levels in S. aureus are extremely low and inconsistent in the literature. To resolve this conundrum, we identified the three-component LTA glycosylation system. In this system, the glycosyltransferase CsbB first charges an undecaprenol phosphate lipid carrier with N-acetylglucosamine in the cytoplasm. This glycolipid is then flipped to the outer leaflet of the cell membrane by the flippase GtcA, where it is available as a substrate for a second glycosyltransferase YfhO to glycosylate LTA. We demonstrate that with the pathway highly expressed, purified LTA contained higher levels of glycosylation and conversely, knocking out the glycosyltransferases eliminated LTA glycosylation. Moreover, we show that D-alanylation competes for receptor modification and by abolishing it, that alone is sufficient to elevate glycosylation levels. Further examination of the genes involved revealed a potential role of LTA glycosylation in stress responses. By exposing the cells to different stresses, we demonstrate that LTA becomes highly glycosylated under high salt conditions. The discovery of the LTA glycosylation mechanism paved the way for identifying the physiological roles and importance of this modification. First, however, we had to circumvent the high levels of D-alanylation which impedes these studies. Complete elimination of D-alanylation is pleotropic thus we also adapted a knockdown system. We show that under standard laboratory conditions, many of the phenotypes associated with LTA is conferred mainly by D-alanylation whereas loss of LTA glycosylation has much more subtle effects on the cell envelope. Furthermore, we investigated the transcription of csbB, revealing complex regulation of this first gene in LTA glycosylation with numerous transcription start sites as both a monocistronic and a bicistronic operon. Using beta-galactosidase reporters, we show that csbB is indeed regulated by the stress-responsive SigB. These results suggest that LTA glycosylation has implications on the cell envelope under stress conditions.