Table 1.
Bacterial strains used in this study.
Table 2.
Sequences of the primers used for detection of surfactant proteins (A; B; C; D), RT-PCR analysis.
Table 3.
Molecular weights of the surfactant proteins and specific antibodies used for their detection in Western blot analysis and Immunofluorecence Micorscopy.
Figure 1.
PCR and RT-PCR analysis of bacterial strains.
A) RT–PCR analysis for transcripts encoding surfactant proteins (A, B, C, D) from S. aureus displaying the samples from different strains; 1. SA 113 [ATCC 35556] aerobe, 2. SA 113 [ATCC 35556] anaerobe, 3. SA N315 [ATCC 12228] aerobe, 4. SA [NCTC 8325] aerobe. B) RT–PCR analysis for transcripts encoding surfactant proteins (A, B, C, D) from P. aeruginosa displaying the samples from different strains; 1. PA 01 aerobe, 2. PA 01 anaerobe, 3. PA [ATCC 14442] aerobe, 4. PA [ATCC 27853] aerobe. C) PCR analysis for genomic DNA from S. aureus and P. aeruginosa displaying the samples from different strains; 1. PA 01 Laboratory strain; 2. PA 154 Environmental isolate, 3. PA (12) Patient isolate, 4. SA 113 [ATCC 35556] Laboratory strain, 5. SA (8) Patient isolate, 6. SA (16) Patient isolate. D) PCR analysis of bacterial DNA from (1) E. coli BL21 (no plasmid) and from (2) E. coli [ATCC 35218] (plasmid). In each case a (RT-)PCR using ß-actin (human), GyrA (bacterial) was performed to include/exclude bacterial/human contamination. (+) indicates the internal positive control for the PCR experiments. E) PCR analysis of bacterial DNA from (1) Pyrococcus furiosus that serves as negative control.
Figure 2.
Multi imaging of S. aureus and P. aeruginosa by means of immunofluorescence and immunogold electron microscopy.
Detection of SP-A, -B, -C and –D by means of immunofluorescence (IHC) and immunogold electron microscopy (EM) for S. aureus (A) and P. aeruginosa (B). The electron microscopically images reveal positive antibody reactivity indicated by the black dots surrounding the bacterial cells. Some of them are marked by using red arrows. In both bacterial strains the antibody reactivity is recognizable mainly on the surface of the microorganisms but also in the cytosol at least to some extend (cf. (D)). Figure D shows cytosolic labeling within the 50 nm ultrathin sections after decreasing the intensity of the background. In case of S. aureus (A) this result is additionally enhanced by the immunofluorescence images (green staining). As proof for unspecific binding of the used antibodies, bacteria were treated and incubated with secondary antibody only (cf. (C)). Neither S. aureus nor P. aeruginosa revealed any reactivity with the antibody.
Figure 3.
Western Blot analysis of bacterial surfactant proteins using monoclonal antibodies originated against human SP-A, -B, -C and -D.
Western blot analysis after SDS gel electrophoresis of bacterial proteins from S. aureus (SA) and P. aeruginosa (PA). After cultivation the proteins were extracted from the bacteria. All investigated samples show distinct bands at the known molecular weights for human surfactant proteins. Lung tissue (Lu) was used as positive control and shows in each case the expected bands for the respective surfactant proteins. ß-actin, a human protein absent in bacteria, was used as a negative control and shows no band.
Table 4.
Protein concentration in ng/mg total protein.
Figure 4.
ELISA quantification of surfactant proteins A, B, C and D in S. aureus (A) and P. aeruginosa (B).
ELISA quantification of SP-A, -B, -C and –D in S. aureus (A) and P. aeruginosa (B) after cultivation using different media conditions (aerobic and anaerobic). For S. aureus (A) the concentration of each surfactant proteins is significantly increased in case of anaerobic cultivation (significance in p is shown in the figure for each surfactant protein. P. aeruginosa also reveals a rise in protein concentration after anaerobic cultivation, but this is not significant.