Characterization of N-terminal Structure of TLR2-activating Lipoprotein in Staphylococcus aureus

Abstract

Staphylococcus aureus is known to activate mammalian immune cells through Toll-like receptor 2 (TLR2).We recently demonstrated that a lipoprotein fraction obtained from S. aureus by Triton X-114 phase partitioning is a potent activator of TLR2. In this study, we separated TLR2-activating lipoproteins expressed in S. aureus and characterized an N-terminal structure. The lipoprotein fraction of S. aureus was prepared by glass bead disruption followed by Triton X-114 phase partitioning. The TLR2-activating molecules were mainly detected in the mass range of 30 -35 kDa. Seven lipoproteins were identified by the mass spectra of their tryptic digests. Among them, three lipoproteins were separated by preparative SDS-PAGE and proved to activate TLR2. After digestion with trypsin in the presence of sodium deoxycholate, the N terminus of the lipopeptide was isolated from lipoprotein SAOUHSC_02699 by normal phase high pressure liquid chromatography and characterized as an S-(diacyloxypropyl)cystein-containing peptide using tandem mass spectra. The synthetic lipopeptide counterpart also stimulated the cells via TLR2. These results showed that the diacylated lipoprotein from S. aureus acts as a TLR2 ligand in mammalian cells.Bacterial infection is one of the major causes of death. Staphylococcus aureus, the most common Gram-positive pathogen, is a major source of mortality in medical facilities (1). The pathogen causes various infectious diseases, including sepsis, endocarditis, and pneumonia. During the infection, S. aureus activates cells and evokes serious inflammation in the host. TLR2 2 has been shown to play a crucial role in the host response to S. aureus (2). However, a detailed understanding of the molecular components that interact with TLR2 in S. aureus cells has not yet been obtained. One of the reported TLR2 ligands was peptidoglycan (PGN) (3), a cell wall component of most bacteria. However, Travassos et al. (4) recently showed that PGN from several bacteria that were highly purified by removal of lipoproteins or lipoteichoic acid (LTA) were not detected by TLR2. Moreover, the minimal active components of the PGN, muramyl dipeptide and desmuramyl dipeptide (␥-D-glutamyl diaminopimelic acid), were determined to be ligands of the intracellular innate immune receptor Nod2/ Nod1 (5, 6), suggesting that PGN is not a ligand of TLR2. Another candidate of TLR2-activating ligands is LTA, a cell surface glycoconjugate of Gram-positive bacteria (3). Morath et al. (7) reported that LTA from S. aureus is a potent stimulator of cytokine release, whereas our group demonstrated that LTA from enterococci, also a major Gram-positive pathogen, has no cytokine-producing activity (8, 9). Furthermore, Han et al. (10) showed that LTA from pneumococci is 100-fold less potent than staphylococcal LTA. These observations suggested that LTA is not a common ligand of TLR2 in Gram-positive pathogens.We also found that the enterococcal LTA fraction contains some contaminants other than LTA and that these components activate immune cells t...

Environment-Mediated Accumulation of Diacyl Lipoproteins over Their Triacyl Counterparts in Staphylococcus aureus

“…The asymmetric distribution of fatty acids at the sn-1 and sn-2 positions of SitC lipoprotein was also confirmed in other S. aureus strains, including strains RN4220 and SA113, and in other lipoproteins, including lipoproteins SA1659 and SA2202 (data not shown). In contrast to our results, Tawaratsumida et al reported a single diacyl structure of an SA2202 lipoprotein modified with a 32:0 fatty acid, in which both the sn-1 and sn-2 positions are modified by the 16:0 fatty acid, from S. aureus SA113 cells grown in BHI medium for 6 h at 37°C (35). Apparently, their ␣-aminoacylation-free diacyl structure can be explained by their culturing conditions for the bacteria, such as the post-logarithmic-growth phase under acidic conditions as revealed in this study.…”

“…However, there are marked discrepancies between their study and ours. In fact, they detected only the diacyl form but not the triacyl form (35). Also, they iso- lated only one S-dipalmitoylglyceryl form rather than a series of diacyl and triacyl lipopeptide forms with different length fatty acids in the sn-1 and a 15:0 fatty acid in the sn-2 positions.…”

N -Acylation of Lipoproteins: Not When Sour

“…They showed that lipoprotein SitC was triacylated when Staphylococcus aureus was in the exponential-growth phase at neutral pH and diacylated in the post-exponential phase at low pH. This result explains the apparently contradictory findings reported in two papers published in 2009 in which Tawaratsumida et al demonstrated that one lipoprotein was diacylated whereas Kurokawa et al showed that another protein was triacylated (8,12). In their study, Kurokawa et al showed that acidic pH is not sufficient for the accumulation of diacylated proteins and that protein synthesis is also required.…”

Lipoproteins in Staphylococcus aureus Mediate Inflammation by TLR2 and Iron-Dependent Growth In Vivo

“…While in Gram-negative bacteria Lpp are further modified at the diacylglyceryl-cysteine by N-acyltransferase (Lnt), a homolog of Lnt was not identified in S. aureus, suggesting diacylated Lpp. The N-terminal part of an isolated staphylococcal Lpp was characterized as diacylated protein (9), whereas another study found evidence for triacylation of the Lpp SitC by a yet unknown enzyme (10).…”