Physiological roles of lipoproteins and their lipidation
In E. coli, 90% of more than 100 putative lipoproteins are expected to localize to the inner leaflet of the outer membrane . Most outer membrane lipoproteins play important roles in producing and maintaining the outer membrane in Gram-negative diderms. Among them, three lipoproteins, BamD (YfiO) [110, 111], LolB  and LptE (RlpB), are essential for bacterial cell growth. BamD is a component of the BamABCDE complex involved in β-barrel protein insertion into the outer membrane . LolB functions in lipoprotein localization, as described above. LptE complexed with LptD (Imp) is involved in transporting lipopolysaccharide to the cell surface . The essentiality of lipid modification and the Lol system in E. coli may be a result of the lethal effects of mislocalization of these essential outer membrane lipoproteins.
Recently, two outer membrane lipoproteins were shown to activate peptidoglycan synthesis in E. coli [114, 115]. At the final stage of peptidoglycan synthesis, penicillin-binding proteins (PBPs) polymerize and cross-link monomeric units of peptidoglycan precursors into a mature peptidoglycan sacculus. Two lipoproteins, LpoA and LpoB, form a complex with PBP1a and PBP1b, respectively, to activate peptidoglycan synthesis. Braun's lipoprotein (Lpp) maintains connections between the outer membrane and peptidoglycan, which stabilizes the outer membrane envelope [116, 117]. Pal is also an abundant outer membrane lipoprotein in E. coli and is involved in outer membrane invagination during the process of cell division .
By contrast, lipoproteins in Gram-positive monoderm bacteria are localized on the outer surface of the cytoplasmic membrane or secreted into the extracellular milieu. Orthologues for ABC transporter periplasmic substrate-binding proteins that are not lipoproteins in Gram-negative bacteria are found as lipoproteins in Gram-positive bacteria [119, 120]. These solute-binding lipoproteins are the most abundant lipoproteins in Gram-positive bacteria . A recent review explains the function of such solute-binding lipoproteins in staphylococcal iron acquisition . Other functional groups of lipoproteins in Gram-positive bacteria, such as β-lactamase for antibiotic resistance, adhesins, and PrsA for protein folding and secretion, have been noted and reviewed previously [12, 13, 120]. Interestingly, B. subtilis PrsA is the only known essential lipoprotein for cell growth ; however, the lipidation enzymes Lgt and Lsp are dispensable. Thus, lipid modification of PrsA is not essential for its function. In M. tuberculosis, seven lipoproteins are essential for cell growth . Of these, LpqW is involved in the synthesis of lipoarabinomannan, a major cell wall component that functions as a virulence factor in this bacterium .
Lipoproteins as TLR2 ligands
Some lipoproteins of pathogenic bacteria are known to interact with host molecules and function as virulence factors against hosts. In addition, the role of lipidation of lipoproteins in inflammation and bacterial pathogenesis has been evaluated using bacteria with mutations affecting lipoprotein biosynthesis. Because the roles of lipoproteins in virulence have been reviewed previously [12, 13, 124, 125], we focus on the TLR2 stimulation abilities of bacterial lipoproteins by new lipidation structures.
Mammalian TLRs recognize specific pathogen-associated molecular patterns derived from various microorganisms, including bacteria, viruses, protozoa and fungi . TLRs induce inflammatory cytokine secretion for host innate immune defence, and are also involved in the establishment of adaptive immunity [7, 126]. Among them, TLR2 plays a major role in the recognition of Gram-positive bacteria . To date, many TLR2 ligand molecules have been suggested, such as lipoproteins, lipopeptides, peptidoglycan, lipoteichoic acid, lipomannans and lipoarabinomannans , whereas other TLRs essentially recognize a single class of pathogen-associated molecular pattern molecules, such as lipopolysaccharide for TLR4. Because these different TLR2 ligands show significant variations in their structures, it is questionable whether TLR2 really interacts with all these suggested ligands . Recent biochemical studies using cell wall component-deficient mutant bacteria clearly demonstrated that bacterial lipoproteins but not lipoteichoic acid or peptidoglycan act as a real native TLR2 ligand molecules [19, 129, 130]. This is supported by analyses of crystal structures of a TLR2 ectodomain in complex with a lipopeptide as described below [131, 132], and by studies using lgt mutants of bacteria such as S. aureus [19, 37, 129, 130, 133], L. monocytogenes  and group B Streptococcus , and using an lipoteichoic acid-deficient S. aureus ltaS mutant [19, 129, 130]. Therefore, only the lipoproteins are real TLR2 ligands, and others may contain lipoproteins as contaminants during their preparations.
TLR2 is unique in its ability to form heterodimer complexes with TLR1 or TLR6. Triacyl and diacyl synthetic lipopeptides, such as N-palmitoyl-S-dipalmitoylglyceryl CSK4 and MALP-2, have been used as TLR2/1 and TLR2/6 agonists, respectively, leading to a model in which triacylated lipopeptides/lipoproteins activate through the TLR2/1 heterodimer, whereas diacylated lipopeptides/lipoproteins activate through the TLR2/6 heterodimer [135-137]. Recently published crystal structures of TLR2/1 and TLR2/6 heterodimerized with a synthetic triacyl and diacyl lipopeptide, respectively, demonstrated that TLR2 interacts with the O-esterified fatty acids, the glyceryl group and the thioether moiety of the S-glycerylcysteine residue of triacyl and diacyl lipopeptides, and that TLR1 recognizes the amide-linked fatty acid of the triacyl form via its hydrophobic cavity, whereas TLR6 does not have such a cavity. However, other biochemical and cell biological studies did not match this model [19, 138-140]. For example, native triacylated SitC lipoprotein purified from S. aureus cells stimulated immune cells via both the TLR2/1 and TLR2/6 heterodimers [19, 138-140]. These studies demonstrate that not only the lipidation structure, but also the amino acid sequence after the lipidated cysteine residue affects the selectivity of TLR2 heterodimers.
How are immune cells stimulated by the three new lipid-modified structures: the lyso, N-acetyl and peptidyl forms? The question of whether or not lyso form lipoproteins that lose one of two esterified fatty acids are recognized by TLR2 heterodimers is an intriguing one. Interestingly, a synthetic lyso form lipopeptide was inactive against TLR2-expressing cells  or TLR2/6-expressing cells, although it activated TLR2/1 expressing cells . However, unexpectedly, two native lyso form lipoproteins stimulate mouse thioglycolate-induced peritoneal macrophages. Additionally, the TLR2 heterodimer selectivities of two different lyso form lipoproteins, namely B. cereus OppA or E. faecalis PrsA, are different from each other: B. cereus OppA stimulates immune cells via both the TLR2/1 and TLR2/6 heterodimers, whereas E. faecalis PnrA mediates the immune signal via the TLR2/6 heterodimer . Therefore, the unusual lyso form lipoproteins can function as TLR2 ligands, and TLR2 heterodimer selectivity may determined not only by the position of the acyl chains, but also by protein sequences and/or lipid compositions (Table 1). Because the lyso form synthetic peptide was not active in TLR2-expressing cells , it can be assumed that the TLR2/1 and TLR2/6 heterodimers (but not the TLR2 homodimer) detect the lyso form structure: in other words, the acquisition of TLR1 and TLR6 via the duplication of the gene for TLR2  might enable host organisms to sense lyso form producing bacteria. Further analyses of structure–function relationships using synthetic lyso form lipopeptides with a variety of different peptide sequences and/or with different fatty acyl groups are required to solve the question of how TLR2 heterodimers recognize the bacterial lyso form lipoprotein at the molecular level.
The TLR2 stimulation ability of another new bacterial lipidation structure, the N-acetyl form, was also examined using mouse macrophages. As expected, the native N-acetyl form lipoproteins and a synthetic N-acetyl form lipopeptide stimulated peritoneal macrophages via TLR2/6 , which is consistent with a previous study that used a synthetic N-acetyl form lipopeptide . Peptidyl form lipoproteins were not examined because of difficulties in obtaining sufficient amounts of native lipoprotein for experiments .
Because typical Gram-positive bacteria are surrounded by a thick cell wall, their lipoproteins are embedded under the wall, and thus they appear to be protected from any physical interaction with the host cell surface TLR2. The in vitro TLR2 stimulation activity of crude peptidoglycan fraction of staphylococci was improved by enzymatic degradation of the peptidoglycan . In the innate immune response, macrophages engulf invading bacteria and deliver them to the phagosome for professional degradation where the TLR complexes are recruited [144-146]. Recent studies have reported that phagocytosis of S. aureus cells together with the subsequent degradation of the cell wall in acidic phagosomes is important for an efficient lipoprotein/TLR2 interaction [147, 148]. Moreover, a fluorescent-labelled purified SitC lipoprotein from S. aureus induced the intracellular accumulation of TLR2 in primary murine keratinocytes, and SitC colocalized with TLR2 but not with TLR4 or nucleotide-binding oligomerization domain 2 (Nod2) .
Watanabe et al.  demonstrated that TLR2 is not involved in the elimination but, instead, the prolonged intracellular survival of S. aureus in macrophages. This intracellular survival has been explained by the TLR2-dependent inhibition of superoxide production, which is augmented by another bacterial cell wall component, d-alanylated wall teichoic acid . Interestingly, another study has reported that the lipoprotein-dependent intracellular survival of S. aureus requires TLR2 but not myeloid differentiation primary response gene 88 (MyD88) .
Because an acidic pH results in S. aureus lipoproteins of the diacyl form , it will be interesting to determine whether S. aureus accumulates diacylated lipoproteins in acidic phagosomes and whether the diacylation is relevant to the immune cell activation and/or intracellular survival of S. aureus.