Susceptibility of chicken embryos to group A streptococci: Correlation with fibrinogen binding

Abstract

One problem in investigating group A streptococcal infections and virulence is the lack of appropriate in vivo models. In this study we introduce the chicken embryo model for determining virulence of Streptococcus pyogenes. We found that M protein positive strains, if administered intravenously, were highly virulent for 12-day-old chicken embryos. The LD50 of the strains tested could be correlated directly with the amount of cell wall exposed M protein, which has been determined by the capacity of streptococci to bind fibrinogen and by the ability of streptococci to survive in fresh normal human blood. The number of colony forming units (cfu) of M+ strains necessary to kill 50% of embryonated eggs was significantly lower (< 10(2) cfu) than for M-variants (> 10(4) cfu). Albumin and/or IgG binding streptococcal cells, which can also take place in proteins of the M protein family which do not bind to fibrinogen, did not show that clear correlation to the virulence in chicken embryos that did fibrinogen binding. Application of anti-streptococcal M protein antisera from chicken and rabbit reduced the lethality of the chicken embryos. In contrast, no correlation was found between lethality of chicken embryos and the in vitro production of erythrogenic toxins by the administered strains. Thus the results indicate that the presence of M-protein with its fibrinogen binding activity on the streptococcal cell surface is necessary for virulence of group A streptococci in the chicken embryo model.

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

“…The binding of human plasma proteins to MC or peptide fragments of MC was investigated after immobilisation of the streptococcal-derived proteins onto nitrocellulose membranes by Western blotting. The membranes were blocked with defatted skimmed milk and then directly incubated with HRPO-labelled human proteins for 4 h. The bacterial dot blot assay to examine binding of human proteins to streptococcal cells was performed as described recently [29,33]. Blots were visualised with 3-amino-9-ethylcarbazole [32].…”

“…inoculation of 100 Wl of di¡erent numbers of streptococci as described recently [29,33]. Before infection, with the help of a counting chamber the bacteria from a 5-ml culture, incubated for 3 h at 37³C in Todd-Hewitt broth, were adjusted to 4U10 3 colony forming units (CFU) per ml with Todd-Hewitt broth, which was diluted with the same volume of PBS [29]. For the protective assay with antiserum, two 3-ml aliquots of the adjusted streptococcal culture were sedimented by centrifugation.…”

“…Recent experiments revealed that such strains of GAS which bind ¢brinogen and/or albumin at their cell surface mostly express proteins of the M family [29,31^33]. Fibrinogen binding seems to be a property of most M proteins [1,7,25,303 2,34] and contributes to the antiphagocytic properties of these cell surface proteins [29,33,42,43]. In our binding experiments with human isolates of GCS and GGS we found that such strains showed complex multiple binding to a test set of plasma proteins comparable to that of GAS [29].…”

“…Fibrinogen binding seems to be a property of most M proteins [1,7,25,303 2,34] and contributes to the antiphagocytic properties of these cell surface proteins [29,33,42,43]. In our binding experiments with human isolates of GCS and GGS we found that such strains showed complex multiple binding to a test set of plasma proteins comparable to that of GAS [29]. For further investigations we selected the group C strain 25287, a wound isolate, which expressed at the cell surface multiple binding to the plasma proteins ¢brinogen, human serum albumin, plasminogen, IgG, IgA, and to vitronectin (not shown).…”

“…In the previously described chicken embryo model it was demonstrated that M proteins contribute to the virulence of GAS for chicken embryos [29,33]. We used this in vivo phagocytosis model to test the GCS strain 25287 with the assumption that the MC may act as virulence antigen like M proteins of GAS.…”