Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation

Lipoproteins are a defining feature of extracellular bacterial membrane surfaces. Acylation of an invariant N-terminal cysteine residue tethers proteins to the cell surface, where they participate in many cellular processes occurring at the membrane-environment interface. Lipoproteins also double as key ligands for bacterial detection by the mammalian innate immune system, which when bound by Toll-like receptor 2 (TLR2) complexes orchestrate a pro-inflammatory response to clear infections. While nearly all bacteria synthesize lipoproteins, variable acylation states can attenuate TLR2 signaling. In Bacillus subtilis, lipoproteins are N-acetylated to form acetylated lipoproteins (Ac-LP) after being transported across the membrane. How high-energy acetyl donors are shuttled to the cell surface for trasnfer is unknown. Using a transposon-based genetic screen to identify mutants with altered TLR2 activation and in vitro reconstitution of the enzyme machinery, we now describe an acetylated heptaprenylglyceryl (Ac-HepG) carrier synthesized by PcrB/YvoF in the cytosol that is required for lipoprotein acetylation. We propose Ac-HepG is shuttled to the outer membrane leaflet for use by the previously uncharacterized integral membrane protein, lipoprotein heptaprenylglyceryl N-acetyl transferase (LhaT) (formerly YpjA), for N-acetylation of the α-amino termini of lipoprotein substrates. We provide evidence that LhaT, which in most Bacillus spp. makes the high-affinity Ac-LP TLR2 ligand, has become pseudogenized in the Bacillus cereus group subclade that harbors opportunistic pathogens. A naturally occurring poly-tyrosine insertion within LhaT from Bacillus anthracis isolates prevents acetyl transfer, allowing the flanking lipoprotein remodeling gene lit to quantitatively convert the population to the TLR2-silent lyso-lipoprotein (lyso-LP) chemotype.