stained with 0.5% crystal violet. Cell morphology was observed wet with an inverted microscope (model Leica DM IRB) equipped with a Leica DC200 digital camera (Leica Microsystems Wetzlar GmbH, Germany).
7.2.5 Cell Adhesion Assay
Cell adhesion assays were performed in 96-well plates coated with fibronectin as described in Section 2.5. Kératinocytes in suspension were pre incubated with 0.01%, 0.1%, 1%, 5%, or 25% concentration of S. aureus culture supernatant diluted in serum-free medium (SFM) for 30 min at room temperature before plating. The cells, in the presence of the S. aureus culture supernatant, were allowed to settle and attach for 2 or 24 hours at 37°C before gentle washing twice with PBS. The number of attached cells in each well was quantified by the MTS assay, and presented as percent of the control treatment in Todd Hewitt broth.
7.2.6 Measurement of the Influx of Propidium Iodide into S. aureus
Culture Supernatant-treated Kératinocytes by Flow Cytometry
Because the intact membrane of live cells excludes a variety of charged dyes such as propidium iodide and trypan blue. Incubation with these dyes results in selective labeling of damaged or dead cells, while live cells show no, or minimal, dye uptake. After crossing the plasma membrane, propidium iodide binds to DNA and
dsRNA, and fluoresces (red) intensely. Subconfluent keratinocyte cultures were harvested and resuspended in fresh KGM. Suspensions of NHK and UP cells were aliquoted at approximately 500,000 cells per tube. The cells were washed once with SFM containing 0.5% BSA (SFM-0.5%BSA) before incubation with 0.01%, 0.1%, 1%, 5%, or 25% concentration of S. aureus culture supernatant diluted in SFM- 0.5%BSA for 30 min at room temperature. After 30 min, cells were washed once with and resuspended in 10%FCS-PBS. Propidium iodide was added in each tube at a final concentration of 1 0 pg/ml, and the cells were left at room temperature for
another 1 0 min before analysis by flow cytometry.
7.3 Results
7.3.1 Hemolytic Activities of Culture Supernatants from a-toxin Producing Strain 8325-4 and a-toxin Knockout Strain DU1090
Lysis of rabbit and sheep erythrocytes by & aureus culture supernatants was used to measure the relative amount of a - and P-toxin present in the supernatants respectively. The results are given in Figure 7-1. The hemolytic activity with sheep erythrocytes from both wild-type a-toxin producing strain 8325-4, and isogenic a - toxin deficient strain DU1090, was similar. This indicates that the production of p- toxin is not affected by the defective gene for a-toxin in DU 1090. Hemolytic titers for a-toxin from strains 8325-4 and DU1090 were 2,048 and 256 respectively. Comparison of the hemolytic titers with a similar study by Nilsson et al (1999) demonstrated a hemolytic titer from DU1090 of 64, the higher hemolytic activity (256) seen in the present preparation may be due to additive effects of other toxins and proteases in the supernatant. It is unlikely to be caused by recovery of a-toxin production by the mutant. This was confirmed by the absence of an approximately 34 kDa protein band from the SDS-PAGE which was present in 8325-4 culture
supernatant (Figure 7-2). The band was identified to be staphylococcal a-toxin by MALDI-TOF MS. The 37 kDa protein band present in DU 1090 culture supernatant was identified to be staphylococcal p-toxin.
positive control | negative control
Control
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D U 1090 (a)( 0 0 0 0 0 0 0 0 0 ^
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DU 1090
(b) Hemolytic titer S. aureusstrain a-toxin P-toxin8325-4 2048 128
DU 1090 256 256
Figure 7-1 In vitro hemolysin production by Staphylococcus aureus strains 8325-4 and DU 1090. The stationary phase culture supernatant at 24 hours from each strain was titered for hemolytic activity against rabbit (a) or sheep (b) erythrocytes. The hemolytic titer is the highest dilution giving rise to lysis of rabbit (a-toxin) or sheep (P-toxin) erythrocytes. Control wells were incubated with water (positive control, five wells on the left) or PBS (negative control, five wells on the right).
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Figure 7-2 S D S -P A G E an alysis o f secreted proteins produced by stap h ylococcal a - toxin -p rod ucin g strain 8 3 2 5 -4 (lane 2) and a -to x in -d e fic ie n t mutant DU 1090 (lane 1 ) at 18 hours stationary phase o f grow th in Todd H ew itt broth. Both lanes contained exo-p rotein s from equal volu m e o f the culture supernatants. The m olecular m asses o f protein standards (lane M , in k ilodaltons) are show n. The protein bands a and b w ere cut and identified to be stap h ylococcal a -to x in and p -toxin resp ectively by M A L D I- TOF m ass spectrom etry.
7.3.2 Comparison of Cell Morphology Changes and Cytotoxicity by Culture Supernatants from a-toxin Producing Strain 8325-4 with a-toxin Knockout Strain DU1090
Figure 7-3 (left column) shows that 5% and 25% 8325-4 culture supernatants were cytotoxic to UP kératinocytes, as demonstrated by cell shrinkage, rounding and detachment. These changes were observed as early as 3 hours. The slight changes of cell morphology observed at 3 hours with 1% 8325-4 culture supernatant were not observed at 24 hours (data not shown).
At concentrations < 5%, culture supernatant from DU 1090 was not cytotoxic to UP cells. Figure 7-3 (right column) shows that, at doses < 5% concentration, no cell shrinkage or rounding was observed. However, membrane rupture and cell detachment were observed after exposure to 25% culture supernatant, as
characterized by nonstained “phantom-like” cells. A similar effect was seen when relative cell viability was determined.
Strain 8325-4 Strain DU 1090
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