Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells - PubMed (original) (raw)

Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells

Tanja Vollmer et al. BMC Microbiol. 2010.

Abstract

Background: Streptococcus gallolyticus subsp. gallolyticus is an important causative agent of infective endocarditis (IE) but the knowledge on virulence factors is limited and the pathogenesis of the infection is poorly understood. In the present study, we established an experimental in vitro IE cell culture model using EA.hy926 and HUVEC cells to investigate the adhesion and invasion characteristics of 23 Streptococcus gallolyticus subsp. gallolyticus strains from different origins (human IE-derived isolates, other human clinical isolates, animal isolates). Adhesion to eight components of the extracellular matrix (ECM) and the ability to form biofilms in vitro was examined in order to reveal features of S. gallolyticus subsp. gallolyticus endothelial infection. In addition, the strains were analyzed for the presence of the three virulence factors gtf, pilB, and fimB by PCR.

Results: The adherence to and invasion characteristics of the examined S. gallolyticus subsp. gallolyticus strains to the endothelial cell line EA.hy926 differ significantly among themselves. In contrast, the usage of three different in vitro models (EA.hy926 cells, primary endothelial cells (HUVECs), mechanical stretched cells) revealed no differences regarding the adherence to and invasion characteristics of different strains. Adherence to the ECM proteins collagen I, II and IV revealed the highest values, followed by fibrinogen, tenascin and laminin. Moreover, a strong correlation was observed in binding to these proteins by the analyzed strains. All strains show the capability to adhere to polystyrole surfaces and form biofilms. We further confirmed the presence of the genes of two known virulence factors (fimB: all strains, gtf: 19 of 23 strains) and demonstrated the presence of the gene of one new putative virulence factor (pilB: 9 of 23 strains) by PCR.

Conclusion: Our study provides the first description of S. gallolyticus subsp. gallolyticus adhesion and invasion of human endothelial cells, revealing important initial information of strain variability, behaviour and characteristics of this as yet barely analyzed pathogen.

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Figures

Figure 1

Dose response analysis of S. gallolyticus adhesion to and invasion of EA.hy926 cells. (A) Adhesion, (B) Invasion. Cells were incubated with decreasing concentrations of three different S. gallolyticus strains (white triangle: isolate 05950, black dot: isolate 21702, white square: DSM 16831), as described in Material and Methods. Error bars indicate standard deviations, n.d.: not detectable.

Figure 2

Adhesion and invasion characteristics of different S. gallolyticus strains to EA.hy926 cells. Displayed are the factorized adhesion to and invasion characteristics of 23 different S. gallolyticus strains (calculated to 1 × 105 CFU/mL) after 2 h infection of EA.hy926 cells. The dashed vertical line indicates the separation of "common" and "noticeable" relations between adhesion and invasion. Error bars indicate standard deviations. Results of statistical analysis of individual strains are arranged in tabular form.

Figure 3

Influence of cell type (EA.hy926/HUVEC) and cell condition (stressed/non-stressed) on the adherence and invasion characteristics of S. gallolyticus. (A) Adhesion to and invasion of endothelial cell lines EA.hy926 and HUVECs after infection with 1 - 9 × 105 CFU/mL of different S. gallolyticus strains. (B) S. gallolyticus strain's adhesion to and invasion of EA.hy926 with and without mechanical stretch 24 h prior to infection with 1 - 9 × 105 CFU/mL bacteria. Results were determined after a 2 h exposure followed by additional 2 h incubation in the presence of antibiotics. n.d.: not detectable.

Figure 4

Biofilm formation and adherence of S. gallolyticus strains to immobilized ECM proteins. Scatter plots show the distribution of the eight ECM proteins and biofilm formation for the different strains/isolates. Individual dots represent the mean values for each strain (quadruplicate determination), the solid horizontal line represents the mean values of the different strains for each ECM protein tested. The dotted horizontal line represents the cut-off value for adherence. The dashed vertical line indicates the separation of the biofilm formation assay. vs: versus, ***P < 0.0001, **P < 0.001, black dot: isolate AC 7070, black triangle: isolate AC 1181.

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Interactions between endocarditis-derived Streptococcus gallolyticus subsp. gallolyticus isolates and human endothelial cells - PubMed (original) (raw)