An important step in listeria lipoprotein research.

Over the last 10 years, DNA sequences of more than 600 bacterial species have been deposited in databases and are now available to search any gene, motif, or regulatory sequence of interest. Although genome data are instrumental in phylogenetic analysis and in silico design of metabolic and regulatory networks, only a very small fraction of the information has been experimentally validated. A striking example is lipoproteins predicted from genome sequences. Despite the predominance of this class of surface proteins in bacteria (up to 0.5 to 8% of the proteome), very few of these proteins have been identified as lipoproteins by biochemical methods (19). In this issue of the Journal of Bacteriology, Baumgärtner et al. (2) report a systematic analysis of lipoproteins of Listeria monocytogenes, a facultative gram-positive intracellular bacterial pathogen that causes severe infections (listeriosis) in both human and animals. These authors used an L. monocytogenes mutant defective in lipoprotein diacylglyceryl transferase (Lgt), an enzyme involved in lipoprotein processing. Three aspects of their study should be highlighted: (i) new findings concerning the roles of Lgt and lipoprotein-specific signal peptidase II (Lsp) during lipoprotein processing (22); (ii) the identification of 26 of the 68 lipoproteins predicted in the initial annotation of the L. monocytogenes strain EGD-e genome (7); and (iii) experimental evidence that a few of these lipoproteins are regulated by PrfA, the master virulence regulator of L. monocytogenes (8). Below, we discuss the significance of these findings separately. Lipoprotein-processing model: differences between grampositive and gram-negative bacteria. Both gram-positive and gram-negative bacteria contain lipoproteins that are a functionally diverse group of surface proteins. The roles assigned to lipoproteins include substrate binding coupled to ABC transport systems, sensing of environmental signals, antibiotic resistance, respiration, germination, conjugation, adherence to and invasion of eukaryotic cells, control of protein secretion and folding, modulation of the immune response, and maintenance of envelope integrity (20). Lipoproteins are synthesized as precursor forms harboring a signal peptide in the N terminus. Upon processing, lipoproteins are ultimately tethered to the membrane via a lipid moiety, diacylglycerol, which is covalently bound to an N-terminal conserved cysteine residue. Work performed with gram-negative bacteria has indicated that the lipidation reaction, carried out by the enzyme lipoprotein diacylglyceryl transferase (Lgt), is followed by cleavage of the signal peptide (22). The enzyme responsible for the latter reaction is the lipoprotein-specific signal peptidase II (Lsp), which recognizes a genuine L 3-S/A 2-A/G 1-C 1 “lipobox.” A further modification step, consisting of addition of an N-acyl moiety to the amino group of the N-terminal cysteine, is carried out by the enzyme N-acyl-transferase (Lnt). The latter modification takes place after cleavage of the signal peptide by Lsp, which leaves the amino group of the cysteine residue free. Thus, the widely accepted model establishes that enzymes involved in prelipoprotein processing act in a tightly Lgt3Lsp3Lnt order (22). Genome analyses of low-G C-content gram-positive bacteria, which include L. monocytogenes, have shown that an lnt gene homolog is not present. Lipoprotein modification in this bacterial group is therefore envisaged as an Lgt3Lsp two-step process (Fig. 1). As Baumgärtner et al. unequivocally demonstrate in their study (2), lack of Lgt activity in L. monocytogenes does not preclude cleavage of nonlipidated prelipoproteins by the signal peptidase Lsp. This conclusion was derived from a proteomic analysis showing that in the lipoproteins released into the extracellular medium by the lgt mutant the C 1 cysteine is the first residue in the N terminus. This protein sequence information demonstrates for the first time that, at least in L. monocytogenes, Lsp is able to cleave signal peptide II at the correct position in nonlipidated prelipoproteins. Interestingly, a recent study performed with a Staphylococcus aureus lgt mutant revealed that the lipoprotein SitC released into the extracellular medium by this mutant has a molecular weight similar to that of the mature SitC lipoprotein present in membrane fractions of wild-type bacteria (18). This observation indicates that Lsp from other gram-positive bacteria may also be able to cleave the signal peptide in nonlipidated prelipoproteins. Baumgärtner et al. also confirmed that the absence of lipid modification in these proteins does not affect the viability of L. monocytogenes, which agrees with previous data reported for lgt mutants of S. aureus, Bacillus subtilis, and Streptococcus pneumoniae (11, 12, 18). Lgt is also a dispensable enzyme for Mycobacterium tuberculosis, although, as has been shown for S. pneumoniae, an Lgt deficiency attenuates virulence (12, 15). Unfortunately, Baumgärtner et al. did not address the role of Lgt in L. monocytogenes virulence using animal models, an aspect that certainly deserves study in the future. Similar to Lgt, the lipoprotein-specific signal peptidase Lsp has been shown to be dispensable for growth in several grampositive bacteria, including L. monocytogenes (6, 13, 21, 23). These findings contrast with those obtained for gram-negative bacteria, in which both Lgt and Lsp are essential enzymes. The presence of two membranes in gram-negative bacteria probably makes accumulation of partially processed prelipoproteins * Corresponding author. Mailing address: Departamento de Biotecnologı́a Microbiana, Centro Nacional de Biotecnologı́a-CSIC, Darwin 3, 28049 Madrid. Spain. Phone: (34) 91 585-4923. Fax: (34) 91 5854506. E-mail: [email protected]. Published ahead of print on 27 October 2006.