Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg.
BACKGROUND
Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA) and cysteine protease (SpeB) as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells.


RESULTS
Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines.


CONCLUSIONS
These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.
Hytönen
Jukka
J
Department of Medical Biochemistry and Molecular Biology, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland. jukka.hytonen@utu.fi
Haataja
Sauli
S
Finne
Jukka
J
D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000502:physiology*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D000078404:Epidermal Cells; D004847:Epithelial Cells / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009061:Mouth Mucosa / Q000166:cytology; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D015534:Trans-Activators / Q000502:physiology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D015534:Trans-Activators; C074779:rgg protein, Streptococcus; D006026:Glycoside Hydrolases; C100181:pullulanase; D003546:Cysteine Endopeptidases; C042477:streptopain