19F NMR studies provide insights into lipid membrane interactions of listeriolysin O, a pore forming toxin from Listeria monocytogenes

Abstract

Listeria monocytogenes is a mammalian pathogen that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised individuals. Its main virulence factor is listeriolysin O (LLO), a pore-forming cholesterol-dependent cytolysin (CDC), which enables bacterial escape from the phagolysosome and contributes to bacterial pathogenicity. Details of cholesterol (Chol) recognition and membrane binding mechanisms by LLO are still not known. Here we used 19F-NMR spectroscopy in order to assess LLO-Chol interactions in solution and in a Chol-rich membrane environment. LLO has six tryptophan residues located in the region of the molecule that is first in contact with lipid membranes. 19F-LLO, which contained 5-fluoro-tryptophans, was prepared by using isotopic labelling in an E. coli expression system. Signals in the 19F-NMR spectrum of 19F-LLO were unambiguously assigned by using a series of single Trp → Phe point mutations. The results employing various cholesterol preparations in solution indicate that tryptophan residues are not directly involved in Chol binding in solution. However, significant chemical shift changes were observed upon LLO binding to Chol-rich membranes, highlighting the role of tryptophan residues in membrane interactions (W512) and oligomerisation (W189 and W489).

More than one way to bind to cholesterol: atypical variants of membrane-binding domain of perfringolysin O selected by ribosome display

“…3) (3b-hydroxy-5-cholestene 3-acetate), which represents a negative control, as it is very similar to cholesterol yet CDCs do not bind to it due to its changed 3b-hydroxyl group. 17,18 Altogether, these results are consistent with the ribosome display affinity selection using SUVs and clearly show that the two selected protein variants exhibit cholesterol-dependent binding that is similar to that of the wild-type PFO. Most importantly, as with the wild-type PFO, both variants remain hemolytically active.…”

“…The critical structural elements responsible for the membrane binding and cholesterol-dependent activity of CDCs have been substantially explored. [15][16][17][18][19][20][21] They include undecapeptide loop (458-ECTGLAWEWWR-468 in PFO) and Scheme 1 Ribosome display was developed to select PFO variants for selective binding of cholesterol in lipid membranes. The approach is composed of (1) in vitro transcription and translation of the DNA library, (2) selection of the protein-ribosome-mRNA complex against lipid vesicles, and (3) amplification of the obtained RNAs for the next round of selection.…”

19F NMR Monitoring of Reversible Protein Post‐Translational Modifications: Class D β‐Lactamase Carbamylation and Inhibition

“…There is growing research interest in reversible post‐translational carbamylation in the regulation of protein function (as classically shown for haemoglobin) and stability . 19 F NMR (using labels introduced by post‐translational modification, or by de novo incorporation of 19 F‐labelled amino acids) will enable further detailed mechanistic investigations into protein carbamylation and its regulation by factors including temperature and pH. It should be noted, however, that our studies highlight the importance of establishing that the properties of the wild‐type enzyme are adequately represented by the 19 F‐labelled variant.…”

Inhibitory Effect of Thymoquinone on Listeria monocytogenes ATCC 19115 Biofilm Formation and Virulence Attributes Critical for Human Infection

“…Listeriolysin O (LLO) is an oligomeric pore-forming toxin secreted by L. monocytogenes . Studies have shown that it is a member of the cholesterol-dependent cytolysin family (Kozorog et al, 2018; Lu et al, 2019). As a main virulence factor in Listeria pathogenesis, LLO has profound effects on the escape of L. monocytogenes from host-cell vacuoles (Portman et al, 2017).…”