Lipoprotein N -Acylation in Staphylococcus aureus Is Catalyzed by a Two-Component Acyl Transferase System

Abstract

Bacterial lipoproteins (Lpps) are a class of membrane-associated proteins universally distributed among all bacteria. A characteristic N-terminal cysteine residue that is variably acylated anchors C-terminal globular domains to the extracellular surface, where they serve numerous roles, including in the capture and transport of essential nutrients. Lpps are also ligands for the Toll-like receptor 2 (TLR2) family, a key component of the innate immune system tasked with bacterial recognition. While Lpp function is conserved in all prokaryotes, structural heterogeneity in the N-terminal acylation state is widespread among Firmicutes and can differ between otherwise closely related species. In this study, we identify a novel two-gene system that directs the synthesis of N-acylated Lpps in the commensal and opportunistic pathogen subset of staphylococci. The two genes, which we have named the lipoprotein N-acylation transferase system (Lns), bear no resemblance to previously characterized N-terminal Lpp tailoring enzymes. LnsA (SAOUHSC_00822) is an NlpC/P60 superfamily enzyme, whereas LnsB (SAOHSC_02761) has remote homology to the CAAX protease and bacteriocin-processing enzyme (CPBP) family. Both LnsA and LnsB are together necessary and alone sufficient for N-acylation in Staphylococcus aureus and convert the Lpp chemotype from diacyl to triacyl when heterologously expressed in Listeria monocytogenes. Acquisition of lnsAB decreases TLR2-mediated detection of S. aureus by nearly 10-fold and shifts the activated TLR2 complex from TLR2/6 to TLR2/1. LnsAB thus has a dual role in attenuating TLR2 signaling in addition to a broader role in bacterial cell envelope physiology. IMPORTANCE Although it has long been known that S. aureus forms triacylated Lpps, a lack of homologs to known N-acylation genes found in Gram-negative bacteria has until now precluded identification of the genes responsible for this Lpp modification. Here, we demonstrate N-terminal Lpp acylation and chemotype conversion to the tri-acylated state is directed by a unique acyl transferase system encoded by two noncontiguous staphylococci genes (lnsAB). Since triacylated Lpps stimulate TLR2 more weakly than their diacylated counterparts, Lpp N-acylation is an important TLR2 immunoevasion factor for determining tolerance or nontolerance in niches such as in the skin microbiota. The discovery of the LnsAB system expands the known diversity of Lpp biosynthesis pathways and acyl transfer biochemistry in bacteria, advances our understanding of Lpp structural heterogeneity, and helps differentiate commensal and noncommensal microbiota.

Mutation of lipoprotein processing pathway genelspAor inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

“…4. The lipoprotein N-acylation transferase system (Lns) converts diacyl lipoproteins to triacylated lipoprotein (29). 5.…”

“…Lnt homologs have been identified in high-GC Gram-positive bacteria (27) but not in low-GC Firmicutes. Despite the lack of an apparent Lnt homolog, N-acylated lipoproteins have been identified in S. aureus (28) and recent work has identified two novel non-contiguous genes lnsA and lnsB which catalyse the N-terminal acylation of lipoproteins in S. aureus (29). The membrane metalloprotease Eep and the EcsAB transporter were shown to be involved in the processing and export of linear peptides, including the signal peptide cleaved by LspA in the lipoprotein processing pathway (30)(31)(32).…”

Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis

“…For whatever reason Lit came to exist, it has the advantage over Lnt and the recently discovered LnsAB 38 and AatD 39 , 40 , the only other known amino terminus acylating enzymes, that upon reaction it does not produce a lyso-phospholipid (lyso-PL) as a second product 1 . Lyso-PLs, as the name implies, are potent surfactants that, at high enough concentrations, will effectively punch holes in and eventually solubilise membranes.…”

Lipoproteins in Gram-Positive Bacteria: Abundance, Function, Fitness

“…These observations raise the important question how firmicutes which do not process a Lnt homolog carry out N-acylation of their Lpp. Recently, it was found that in S. aureus two genes/enzymes are involved in N-acylation of Lpp (Gardiner et al, 2020). They are referred to as N-acylation transferase system (Lns).…”