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. 2007 Jan;189(2):313-24.
doi: 10.1128/JB.00976-06. Epub 2006 Oct 13.

Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes

Maja Baumgärtner  1 , Uwe Kärst, Birgit Gerstel, Martin Loessner, Jürgen Wehland, Lothar Jänsch
Affiliations

Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes

Maja Baumgärtner et al. J Bacteriol. 2007 Jan.

Abstract

Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.

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Figures

FIG. 1.
FIG. 1.
In vitro infection analyses. (A) Fluorescence microscopy of mouse fibroblast cell line 3T3 infected (multiplicity of infection, 100) with the EGDe wild-type (wt) or Δlgt strain. At 3 h postinfection, F-actin was stained with phalloidin (green), and bacteria were labeled with anti-Listeria antibodies (red). (B) Invasion and intracellular growth analyses of the EGDe wild-type and Δlgt strains. Human epithelial cells (Caco-2) and mouse fibroblast cell line 3T3 were inoculated with the wild-type and Δlgt strains at a multiplicity of infection of approximately 6. After 1, 3, 5, and 7 h of incubation in the presence of gentamicin, cells were washed with PBS and lysed by addition of cold Triton X-100. Viable bacteria released from the cells were plated onto BHI agar plates to count the colonies. The data graphs the means and standard deviations of triplicate measurements.
FIG. 2.
FIG. 2.
(A) Expression of Lgt in the EGDe wild-type strain (wt), the Δlgt strain, and the complemented Δlgt(pE1lgt) strain. Total cell extracts (15 μg protein) were separated by SDS-PAGE and transferred to a PVDF membrane, and Lgt expression was analyzed with polyclonal anti-Lgt antibodies. (B) Metabolic labeling of lipoproteins with [14C]palmitic acid. The L. monocytogenes EGDe wild-type strain (wt), the Δlgt strain, and the complemented Δlgt(pE1lgt) strain were grown in the presence of [14C]palmitic acid until the late exponential phase. Proteins were extracted with chloroform-methanol, separated by SDS-PAGE, and transferred to a PVDF membrane. 14C-labeled palmitoylated polypetides were analyzed by autoradiography. No labeled protein band was detected in the Δlgt extract.
FIG. 3.
FIG. 3.
(A) Dual-channel image showing a comparison of the extracellular proteomes of the L. monocytogenes EGDe wild-type strain (orange) and the Δlgt strain (blue). Cells of the wild-type and Δlgt strains were grown in BHI medium and harvested after the cultures reached the stationary phase. Following precipitation with trichloroacetic acid-acetone proteins were separated by 2-D gel electrophoresis. The numbers indicate proteins which are detected exclusively in the extracellular proteome of the Δlgt deletion mutant or are upregulated in this mutant. All of these proteins identified by MALDI-TOF mass spectrometry have a typical prolipoprotein signal sequence. The numbers correspond to the numbers in Tables 1 and 2. (B) Dual-channel image showing a comparison of the extracellular proteomes of the EGDe wild-type strain (orange) and the complemented Δlgt(pE1lgt) strain (blue). The wild-type phenotype is completely restored in the Δlgt strain harboring an additional plasmid with the lgt gene.
FIG. 4.
FIG. 4.
Comparison of the extracellular proteomes of the L. monocytogenes Δlgt mutant grown without (A) and with (B) globomycin, which selectively inhibits the lipoprotein-specific signal peptidase II (Lsp). Cells were cultivated and samples were prepared as described in the legend to Fig. 3.
FIG. 5.
FIG. 5.
Growth curves for the L. monocytogenes Δlgt(pES11) (•), ΔprfA/Δlgt(pES11) (▵), and Δlgt(pESP11prfA) (□) strains in minimal medium at 37°C. OD600nm, optical density at 600 nm.
FIG. 6.
FIG. 6.
Comparison of the extracellular proteomes of the L. monocytogenes Δlgt ΔprfA/Δlgt(pES11) (−prfA) and Δlgt(pESP11prfA) (+prfA) strains. Bacteria were grown in minimal medium, and Δlgt(pES11) cells were harvested in the late exponential phase. Cells of the Δlgt(pESP11prfA) strain were harvested at the same time as cells of the Δlgt (pES11) strain at an optical density at 600 nm of approximately 0.4. After precipitation with trichloroacetic acid-acetone, extracellular proteins were separated by 2-D gel electrophoresis as described in Materials and Methods. MALDI-TOF mass spectrometry resulted in identification of five proteins (ActA, InlA, InlC, Lmo2219, and Lmo0366) with significantly increased extracellular expression levels that correlated with increased PrfA expression. The oligopeptide-binding protein OppA was present in two differently regulated protein spots. The observed pI shift suggested that there was PrfA-regulated posttranslational modification of OppA.
FIG. 7.
FIG. 7.
(A) Immunoblot analyses of the extracellular proteomes of the L. monocytogenes EGDe wild-type (wt) and Δlgt strains. Extracellular protein fractions were prepared as described in the legend to Fig. 3, separated by 2-D gel electrophoresis, and blotted on PVDF membranes. The blots were developed using polyclonal antibodies against the predicted lipoproteins Lmo1800 and Lmo2595. (B) Immunoblot analyses of the extracellular proteomes of the L. monocytogenes Δlgt(pES11) (wt prfA), ΔprfA/Δlgt(pES11) (−prfA), and Δlgt(pEPS11prfA) (+prfA) strains. Extracellular protein fractions were prepared as described in the legend to Fig. 6, transferred to PVDF membranes using a slot blot unit, and reacted with polyclonal antibodies against the predicted lipoproteins Lmo1800 and Lmo2595.
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Comment in

  • An important step in listeria lipoprotein research.
    García-del Portillo F, Cossart P. García-del Portillo F, et al. J Bacteriol. 2007 Jan;189(2):294-7. doi: 10.1128/JB.01577-06. Epub 2006 Oct 27. J Bacteriol. 2007. PMID: 17071755 Free PMC article. No abstract available.

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