Synthesis of Lyso-form Lipoproteins and Their Implications in Copper Resistance and the Host Immune Response
Open Access
- Author:
- Armbruster, Krista Marie
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 20, 2019
- Committee Members:
- Timothy Charles Meredith, Dissertation Advisor/Co-Advisor
Sarah Ellen Ades, Committee Chair/Co-Chair
Paul Babitzke, Committee Member
Kathleen Postle, Committee Member
Margherita Teresa-Anna Cantorna, Outside Member - Keywords:
- lipoprotein
Firmicutes
copper resistance
Toll-like receptor
acylation
mass spectrometry - Abstract:
- Ubiquitous in bacteria, lipoproteins are involved in an array of biological processes, including nutrient acquisition, signal transduction, protein folding, and more. While their globular protein domain can vary greatly, all lipoproteins are anchored to the cell membrane by a lipidated N-terminal cysteine residue. How lipoprotein acylation occurs is well-characterized in model Gram-negative bacteria: (i) following insertion into the cytoplasmic membrane by an N-terminal signal peptide, lipoprotein diacylglycerol transferase (Lgt) attaches a diacylglycerol moiety from a neighboring phospholipid to the thiol of the conserved cysteine, (ii) lipoprotein signal peptidase (Lsp) cleaves the signal peptide immediately N-terminal to the cysteine, then (iii) lipoprotein N-acyltransferase (Lnt) attaches a third acyl chain to the exposed α-amino group, completing the mature triacylated lipoprotein. Based on the absence of lnt sequence orthologs in their genomes, lipoproteins of Gram-positive bacteria were assumed to be diacylated. However, studies have demonstrated lipoprotein triacylation in organisms lacking lnt. Additional lipoprotein forms were also discovered in low-GC Firmicutes, named the peptidyl form, the N-acetyl form, and the lyso form, all featuring N-terminal modifications. Characterized by an N-acyl-S-monoacylglyceryl cysteine structure, the lyso form was found in Enterococcus faecalis, Bacillus cereus, Lactobacillus delbrueckii, and Streptococcus sanguinis. Following the lyso form’s discovery, the enzyme(s) responsible for its synthesis, how it is synthesized, and the overall role of N-terminal lipoprotein modification in Gram-positive bacteria, which appears to differ from that of Gram-negative bacteria, remained unknown. This Dissertation has sought to answer each of these questions. Using a complementation rescue assay of a conditional-lethal lnt mutant in Escherichia coli, we identified the enzyme that converts lipoproteins to the lyso form in E. faecalis and B. cereus, named lipoprotein intramolecular transacylase (Lit). Using a combination of microbial genetics, traditional molecular biology approaches, and intensive characterization of lipoproteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), we found that expression of Lit in E. coli converts lipoproteins to the lyso form, thereby allowing studies of Lit and the lyso form in a model system. Deletion of Lit from its native organism abolishes maturation to the lyso form, resulting in diacylglycerol-modified lipoproteins. Discovery of Lit allowed for investigation into its phylogeny, revealing an unexpected distribution of lit in a transmissible operon encoding several copper resistance determinants. This operon is found in environmental isolates of Enterococcus spp. and Listeria monocytogenes, the latter previously only known to elaborate diacylglycerol-modified lipoproteins. Transcriptional and mass spectrometric analyses demonstrated that expression of this Lit ortholog is induced by elevated levels of copper, in turn converting lipoproteins to the lyso form in L. monocytogenes. We propose that copper coordinates with the exposed α-amino group of diacylated lipoproteins at the membrane interface, facilitating entry into the cell and causing cellular stress. Lyso-lipoprotein formation may weaken this coordination and subsequent uptake, suggesting a greater physiological role for N-terminal lipoprotein modifications. We also demonstrated that conversion to the lyso form markedly decreases recognition by Toll-like receptor 2 (TLR2), occurring predominantly through the TLR2/6 heterodimer, providing the first insight into the innate immune response to the lyso form versus its diacylglycerol-modified precursor. It was previously theorized that Lit functions by a novel intramolecular transacylation mechanism, by which an acyl chain from the thiol-bound diacylglycerol moiety is transferred to the α-amino group of the cysteine. To test this hypothesis, an elaborate E. coli mutant was constructed that is able to incorporate exogenous deuterium-labeled fatty acids into its diacylglycerol-modified lipoproteins. Conditions were identified in which membrane-reconstituted Lit appears to be active in vitro. Combining Lit and the labeled diacylated substrate in a reaction, the resulting lyso-form lipoproteins will be analyzed by mass spectrometry. This may reveal a labeled N-acyl chain, which would confirm Lit’s proposed mechanism of action.