In the absence of Lgt, lipoproteins are shed from Streptococcus uberis independently of Lsp

Abstract

The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.

Functional Analyses of Mycobacterial Lipoprotein Diacylglyceryl Transferase and Comparative Secretome Analysis of a Mycobacterial lgt Mutant

“…In M. smegmatis, a total of approximately 140 putative lipoproteins are annotated. This increased release of lipid anchor-lacking lipoproteins in the lgt mutant verifies previous secretome results and confirms the function of Lgt in M. smegmatis (1,3,8,9,43). We also searched the MALDI-TOF data set of the ⌬lgt mutant secretome results for peptide masses corresponding to lipoprotein-specific signal peptides to analyze whether lipoproteins are released in processed or nonprocessed forms.…”

“…The lipid structure is thought to anchor proteins to hydrophobic membranes. In B. subtilis, E. coli, and Listeria, failure to attach the lipid anchor results in the loss of precursor lipoproteins (3,8,9,43). The lack of membrane retention results in the accumulation of these proteins in the culture filtrate.…”

“…In a secretome analysis of B. subtilis, seven lipoproteins were found to be shed into the extracellular medium in the wild type, and more than 20 lipoproteins were released into the medium in large amounts in the B. subtilis lgt mutant (1). Lipoproteins lacking the lipid anchor were reported to be released into the extracellular medium after signal sequence cleavage by Lsp, SpaseI, or alternative proteases or without further processing in Streptococcus species, L. monocytogenes, and B. subtilis, respectively (1,3,8,9,43).…”

The molecular mechanism of bacterial lipoprotein modification—How, when and why?

“…Lgt of S. aureus can complement a temperature-sensitive mutant of E. coli, suggesting that Lgt is functionally conserved among bacterial species (Qi et al, 1995). Deletion mutants of lgt in firmicutes often lead to shedding of lipoproteins into the culture supernatant in non-lipidated precursor form with uncleaved signal peptide (Leskelä et al, 1999) or processed by an unidentified signal peptidase (Stoll et al, 2005;Denham, Ward and Leigh 2009) or Lsp (Baumgärtner et al, 2007;Henneke et al, 2008). Two Lgt homologues were identified in Streptomyces coelicolor, Lgt1 and Lgt2, which can both complement the lgt mutant of S. scabies, suggesting that both enzymes are functional enzymes (Widdick et al, 2011).…”

“…The Lsp enzymes can complement an lsp depletion strain of E. coli only in the absence of Lpp suggesting that Lsps of M. xanthus are less efficient than Lsp of E. coli. Lipoproteins of Streptococcus uberis are processed by Eep, a homologue of enhanced expression of pheromone from Enterococcus faecalis (An, Sulavik and Clewell 1999), in the absence of Lsp in late-stationary-phase cells (Denham et al, 2009).…”