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. 2005 Apr;73(4):2411-23.
doi: 10.1128/IAI.73.4.2411-2423.2005.

Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation

Hartmut Stoll  1 , Jörn Dengjel, Christiane Nerz, Friedrich Götz
Affiliations

Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation

Hartmut Stoll et al. Infect Immun. 2005 Apr.

Abstract

A lipoprotein diacylglyceryl transferase (lgt) deletion mutant of Staphylococcus aureus SA113 was constructed. The lipoprotein and prelipoprotein expression, the growth behavior, and the ability of the mutant to elicit an immune response in various host cells were studied. In the wild type, the majority of [14C]palmitate-labeled lipoproteins were located in the membrane fraction, although some lipoproteins were also present on the cell surface and in the culture supernatant. The lgt mutant completely lacked palmitate-labeled lipoproteins and released high amounts of some unmodified prelipoproteins, e.g., the oligopeptide-binding protein OppA, the peptidyl-prolyl cis-trans isomerase PrsA, and the staphylococcal iron transporter SitC, into the culture supernatant. The growth of the lgt mutant was hardly affected in rich medium but was retarded under nutrient limitation. The lgt mutant and its crude lysate induced much fewer proinflammatory cytokines and chemokines in human monocytic (MonoMac6), epithelial (pulmonary A549), and endothelial (human umbilical vein endothelial) cells than the wild type. However, in whole blood samples, the culture supernatant of the lgt mutant was equal or even superior to the wild-type supernatant in tumor necrosis factor alpha induction. Lipoprotein fractionation experiments provided evidence that a small proportion of the mature lipoproteins are released by the S. aureus wild type despite the lipid anchor and are trapped in part by the cell wall, thereby exposing the immune-activating lipid structure on the cell surface. Bacterial lipoproteins appear to be essential for a complete immune stimulation by gram-positive bacteria.

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Figures

FIG. 1.
FIG. 1.
Allelic replacement of lgt in the genome of S. aureus SA113 (A) and map of the complementation plasmid pRBlgt (B). (A) A plasmid based on the shuttle vector pBT2 that allowed replacement of lgt by ermB through homologous recombination was constructed. The flanking regions of lgt, hprK upstream, and yvoF/yvcD′ downstream were amplified from the S. aureus SA113 genome. ermB contains its own promoter, which mediates moderate expression of downstream genes and does not contain a transcriptional termination signal. The ermB gene in the isogenic lgt::ermB mutant is in the same orientation as lgt in the wild type. The postulated functions of the proteins encoded by the four genes of the operon are indicated. (B) Only the relevant part of the complementation plasmid pRBlgt is shown. The lgt gene with its original initiation translational signal was cloned using the XbaI and SacI restriction sites of the multiple cloning site of the shuttle vector pRB474. Transcription of lgt is driven by the constitutive vegII promoter from B. subtilis. lgt, prolipoprotein diacylglyceryl transferase; P, vegII promoter; T, transcriptional terminators from phage λ (To) and from rrnB of E. coli (T1); cat, chloramphenicol acetyltransferase gene; bla, β-lactamase.
FIG. 2.
FIG. 2.
Lack of lipid modification of lipoproteins in the lgt mutant. Coomassie blue staining of membrane proteins (A) and autoradiograph of membrane proteins in the same gel (B). Wild-type S. aureus, the lgt mutant, and the complemented mutant were cultivated in MHB medium with shaking for 4.5 h at 37°C. Proteins were labeled and isolated as described in Materials and Methods and separated by SDS-PAGE on 12.5% polyacrylamide gels. Right margin, size markers in kilodaltons.
FIG. 3.
FIG. 3.
Cell-wall-associated proteins. Cell-wall-associated lipoproteins were released from whole bacterial cells with 1.5 M LiCl. Bacteria were grown in MHB supplemented with [14C]palmitic acid. Total protein (12 μg) was separated on an SDS-12.5% polyacrylamide gel and stained with Coomassie brilliant blue (A and B). Gels were dried and autoradiographed for 4 or 5 days (C). WT, wild type.
FIG. 4.
FIG. 4.
Release of proteins into the culture supernatant. Proteins from culture supernatants of stationary phase cultures (grown in BM for 14 h with shaking) of S. aureus wild type, the lgt mutant, and the complemented mutant were separated by SDS-PAGE and stained with Coomassie brilliant blue. At least 14 additional or more highly expressed protein bands (arrows) were visually detected in the supernatant of the lgt mutant. Some of the proteins were cut out and partially sequenced. Autolysin (Atl), oligopeptide-binding protein (OppA) and autolysin homolog protein comigrate; an unidentified protein and 1 to 4 homologous proteins comigrate at the 35-kDa position. PrsA, peptidyl-prolyl cis-trans isomerase; SitC, binding protein of staphylococcal iron transporter.
FIG. 5.
FIG. 5.
Localization of SitC to the membranes of S. aureus SA113 (pTXsitC) and S. aureus SA113 lgt::ermP (pTXsitC). Expression of sitC was induced by the addition of xylose after 1 h of growth. Bacteria were grown in BM for 13 h, and proteins from the membrane fraction were separated by SDS-PAGE and stained with Coomassie brilliant blue. pTX16 is the control plasmid. In the wild-type (WT) strain carrying pTXsitC, SitC migrates at the 32-kDa position, whereas in the lgt mutant carrying pTXsitC, SitC migrates at the 34-kDa position, very likely because it contains a leader sequence.
FIG. 6.
FIG. 6.
Growth of S. aureus wild-type and lgt mutant in different growth media. Fresh medium was inoculated to an optical density (OD) at 578 nm of 0.1 with staphylococcal overnight cultures that had been grown for 18 h in the respective medium. (A) BM; (B) RPMI-AV1. Filled squares, wild type; open circles, lgt mutant.
FIG. 7.
FIG. 7.
Production of IL-6, IL-8, or MCP-1 by A549 (A) and HUVEC (B) upon stimulation with S. aureus wild-type and lgt mutant cells. For time kinetic studies, A549 cells were infected with a ratio of 40:1; HUVEC were infected with a ratio of 50:1 to 75:1. Dose-dependent stimulation was determined after 36 h (results not shown). In some experiments, a basal IL-6 or IL-8 secretion was observed. Data represent means ± standard deviations of three different experiments, each carried out in duplicate. Filled circles, wild type; open circles, lgt mutant; gray diamonds, complemented lgt mutant; gray triangles, medium (control).
FIG. 8.
FIG. 8.
Induction of TNF-α or IL-10 in MonoMac 6 cells by S. aureus wild type and the lgt mutant (A through C) or crude extract (D). For time-dependent studies with bacteria, MonoMac 6 cells were infected with a ratio of 40:1. Dose-dependent stimulation was determined after 8 h. Panels A and C show one representative experiment of three, each carried out in duplicate. In panel B, data of three different experiments carried out in duplicate are shown. Panel D illustrates one representative experiment carried out with three separate crude extract preparations. Crude extract was derived from overnight cultures in DMEM-F-12, and the indicated amounts were added to 106 monocytes; 10 μg of the crude extract (wet weight)/ml corresponds to 2.6 × 106 bacteria. Error bars indicate standard deviation. Filled circles, S. aureus SA113 wild type; open circles, the isogenic lgt::erm mutant; gray diamonds, complemented lgt mutant; gray triangles, medium.
FIG. 9.
FIG. 9.
Cytokine induction in human whole blood by S. aureus cells, supernatants, and synthetic lipopeptides. Fresh blood samples were treated for 5 h with either bacteria (A) or bacterial culture supernatants (B through D). (C and D) Synthetic lipopeptides were added to the bacterial supernatants (diluted 50% with medium) at the indicated concentrations prior to stimulation of the blood samples. Values are means of three (A) or four (B) different experiments, each carried out in duplicate. Panels C and D show one representative experiment of three, carried out in triplicate. Error bars indicate standard deviations. Filled circles, S. aureus SA113 wild type; open circles, the isogenic lgt::ermP mutant; gray diamonds, complemented lgt mutant; gray triangles, medium (control).
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