Investigating the Uptake of astexins-2 and -3 through TonB-dependent Transporters

Abstract

Lasso peptides are a class of ribosomally-synthesized and post-translationally modified peptides (RiPPs) with a characteristic threaded lasso structure. Genome mining studies have led to the identification of lasso peptide biosynthetic gene clusters in Asticcacaulis excentricus. One of these gene clusters contain the genes needed for the synthesis, maturation, and function of astexin-2, and astexin-3. The gene cluster of A. excentricus contains the gene encoding AtxF, a putative TonB-dependent transporter (TBDT). TBDTs often act to transport siderophores, small molecules acting as metal ion scavengers. Thus it is hypothesized that astexin-2 and astexin-3 act as siderophores. The purpose of this study is to investigate the uptake mechanism of these lasso peptides through AtxF. Additionally, the gene cluster in A. excentricus also contains the gene encoding a FecR-like protein. FecR homologs have been shown to interact with TBDTs in other organisms and hence uptake patterns of the lasso peptides were investigated in the presence and absence of FecR. Results show that AtxF can take up astexin-2 and astexin-3 from its surroundings. Additionally, FecR co-expression augments AtxF expression levels potentially by stabilizing it through a protein-protein interaction. AtxF and FecR co-expression also enhances uptake of astexin-3 and lowers uptake of astexin-2, indicating that both lasso peptides are taken up through different mechanisms. The effect of metal ions on the uptake of astexin-3 was also explored and results suggested the role of astexin-3 as a ferric ion siderophore as well as a factor to lower metal ion related toxicity at higher metal concentrations. Overall, this study sheds light on the functions of astexin-2 and astexin-3 lasso peptides and their uptake mechanisms. Understanding the uptake of these lasso peptides can potentially lead to novel medical applications in the future such as improved antibiotic target delivery, a major step in fighting against antibiotic resistance.