Virulence of group A streptococci in fertile hens eggs is mainly effected by M protein and streptolysin O

In this study we have investigated whether streptolysin O contributes to th e virulence of group A streptococci. For this purpose we generated M-negati ve and SLO-negative mutants by insertion mutagenesis into the chromosome of an M type 1 strain. The inactivation of M1 protein expression was achieved by the construction of the integrative plasmid pSFABS, which contains the internal fragment abs of the emm1 gene. Integration of pSFABS by homologous recombination into the chromosome of strain 38 541 resulted in the generat ion of mutant EMM1. Inactivation of slo with plasmid pFWSLOD resulted in tw o different mutant forms. The homologous recombination with plasmid pFWSLOD carrying the two slo fragments slo1 (899 base pairs in the 5 ' region) and slo2 (709 base pairs in the downstream part) resulted in mutants SLO3, SLO 4 and SLO17. In SLO17 a double crossover event took place with insertion of the spectinomycin resistance gene aad9 between the slo fragments 1 and 2. In mutants SLO3 and SLO4 the homologous recombination with the same plasmid led to the integration of the whole plasmid construct into the chromosome of strain 38 541. Both forms of mutation failed to express SLO. In mutant S LO4 additionally M1 protein expression was significantly decreased. The mut ants EMM1 (M-, SLO+) and SLO4 (M decreased, SLO-) showed a reduced binding to collagen-coated surfaces. In contrast the mutants SLO3 and SLO17 (both M +, SLO-) and the wild-type strain 38 541 (M+, SLO+) showed an affinity to c ollagen similar to purified M1 protein. All mutants were less virulent for chicken embryos compared to the wild-type strain after infection by intrave nous injection as well as by application onto the chorioallantoic membrane. The results show that besides M protein SLO can also influence virulence o f group A streptococci. Moreover, it became obvious that streptococci need more than one tool to fully develop their infectious potential.