Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c

Abstract

Background: Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid.

Results: We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed.

Conclusions: Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

Figure 1

MALDI-TOF and MALDI-TOF/TOF analysis of the N-terminal peptides of LprF. A. MS analysis of AspN-digested peptides of LprF purified from M. bovis BCG parental strain. Filled triangle, diacylglycerol (C16/C19) + N-acyl (C16) modified and glycosylated N-terminal peptide, open triangle, diacylglycerol (C16/C19) + N-acyl (C19) modified and glycosylated N-terminal peptide B. MS/MS analysis of the N-terminal peptide of LprF from M. bovis BCG parental strain. Eliminated fragments of LprF modifications are shown in the upper part of the spectrum. ➀ Tuberculostearinamide + Didehydroalanine, ➁ Diacylthioglyceryl (C16/C19), ➂ Hexose. C. Schematic drawing of the modified +1 cysteine with the cleavage sites of each identified m/z signal.

Figure 2

A comparison of the genomic region of Lnt homologues in mycobacteria. Black bars/arrows indicate Lnt homologues. A second domain is fused to the lnt domain in M. tuberculosis Rv2051c, and M. bovis BCG BCG_2070c (grey arrows) and is homologous to M. smegmatis MSMEG_3859 (grey arrow). White arrows indicate orientation of surrounding genes.