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. CONCLUSION: 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

Bacterial adhesion of Streptococcus suis to host cells and its inhibition by carbohydrate ligands

Peer reviewe

Use of tetravalent galabiose for inhibition of Streptococcus suis serotype 2 infection in a mouse model

Peer reviewe

Inhibition of Pneumolysin Cytotoxicity by Hydrolysable Tannins

Streptococcus pneumoniae causes invasive infections such as otitis media, pneumonia and meningitis. It produces the pneumolysin (Ply) toxin, which forms a pore onto the host cell membrane and has multiple functions in the pathogenesis of S. pneumoniae. The Ply C-terminal domain 4 mediates binding to membrane cholesterol and induces the formation of pores composed of up to 40 Ply monomers. Ply has a key role in the establishment of nasal colonization, pneumococcal transmission from host to host and pathogenicity. Altogether, 27 hydrolysable tannins were tested for Ply inhibition in a hemolysis assay and a tannin-protein precipitation assay. Pentagalloylglucose (PGG) and gemin A showed nanomolar inhibitory activity. Ply oligomerization on the erythrocyte surface was inhibited with PGG. PGG also inhibited Ply cytotoxicity to A549 human lung epithelial cells. Molecular modelling of Ply interaction with PGG suggests that it binds to the pocket formed by domains 2, 3 and 4. In this study, we reveal the structural features of hydrolysable tannins that are required for interaction with Ply. Monomeric hydrolysable tannins containing three to four flexible galloyl groups have the highest inhibitory power to Ply cytotoxicity and are followed by oligomers. Of the oligomers, macrocyclic and C-glycosidic structures were weaker in their inhibition than the glucopyranose-based oligomers. Accordingly, PGG-type monomers and oligomers might have therapeutic value in the targeting of S. pneumoniae infections

Urea-water-solution properties:density, viscosity, and surface tension in an under-saturated solution

Abstract A temperature-concentration dependent surface fit for the relative viscosity of a urea-water-solution (UWS) is calculated based on experimental and literature data. For the surface fit, a 2D Lorentzian function was used, where the x-axis was assigned to a urea mass fraction and the y-axis to the solution temperature and the rest of the Lorentzian function parameters were optimized based on the experimental and literature data. The surface model describes the relative viscosity of under-saturated urea-water-solution. The experimental data for the kinematic viscosity was measured with an Ubbelohde capillary viscometer whose temperature was controlled with a thermostat. The temperature and concentration range was from 293.15 to 353.15 K in 10-K increments and for urea mass fractions from 0.325 to 0.7. The kinematic viscosity values from the experiment were converted to relative viscosity by calculating the density of the UWS. An exponential fit was calculated to describe the specific gravity of the UWS based on literature data. Additionally, the surface tension of the UWS was measured at room temperature (293.15 K) in a mass fraction range from 0.302 to 0.596. As a result, simple models describing UWS properties were obtained and these models can be implemented into computational fluid dynamics (CFD) simulations

Oligosaccharide-receptor interaction of the Galα1-4Gal binding adhesin of Streptococcus suis : Combining site architecture and characterization of two variant adhesin specificities

The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Galα1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAcβ1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO- 6', HO-2, and HO-3 hydroxyls on the β'α-side of the Galα1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms

Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-alpha 1-4-galactose-containing Glycoconjugates CONVERGENT EVOLUTION OF BACTERIAL PATHOGENS TO BINDING OF THE SAME HOST RECEPTOR

Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-alpha 1-4-galactose (Gal alpha 1-4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Gal alpha 1-4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Gal alpha 1-4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Gal alpha 1-4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO(3) (CD77). The N terminus of SadP was shown to contain a Gal alpha 1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Gal alpha 1-4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure