Within a genomic locus termed the vir regulon, virR genes of opacity factor-nonproducing (OF ؊) group A streptococci (GAS) are known to control the expression of the genes encoding M protein (emm) and C5a peptidase (scpA) and of virR itself. Within the corresponding genomic locus, opacity factor-producing (OF ؉) GAS harbor additional emm-related genes encoding immunoglobulin G-and immunoglobulin A-binding proteins (fcrA and enn, respectively). The virR gene region of the OF ؉ GAS M-type 49 strain CS101 was amplified by PCR, and 2,650 bp were directly sequenced. An open reading frame of 1,599 bp exhibited 76% overall homology to published virR sequences. By utilizing mRNA analysis, the 5 ends of two specific transcripts were mapped 370 and 174 bp upstream of the start codon of this open reading frame. The deduced sequences of the corresponding promoters and their locations differed from those of previously reported virR promoters. Transcripts from wild-type fcrA49, emm49, enn49, and scpA49 genes located downstream of virR49 were characterized as being monocistronic. The transcripts were quantified and mapped for their 5 ends. Subsequently, the virR49 gene was inactivated by specific insertion of a nonreplicative pSF152 vector containing recombinant virR49 sequences. The RNA from the resulting vir-mut strain did not contain transcripts of virR49, fcrA49, emm49, or enn49 and contained reduced amounts of the scpA49 transcript when compared with wild-type RNA. The mRNA control from the streptokinase gene was demonstrated not to be affected. When strain vir-mut was rotated in human blood, it was found to be fully sensitive to phagocytosis by human leukocytes. Thus, the present study provides evidence that virR genes in OF ؉ GAS could be involved in the control of up to five vir regulon genes, and their unaffected regulatory activity is associated with features postulated as crucial for GAS virulence.

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

Group A streptococci (GAS) express up to four types of secreted DNases. Although GAS infections are correlated with the production of anti-DNase B antibodies, the roles of DNases in the pathogenesis of GAS infections remain unclear. From a lambda library of serotype M49 strain CS101 GAS genome, a 2,147-bp fragment expressing DNase activity on an indicator agar was identified and sequenced. One 1,155-bp open reading frame (ORF) was identified in this fragment. This ORF was found to be 48% identical on the amino acid level to group C streptococcal DNase (Sdc). The regions of highest homology corresponded to amino acid residues that were also identified as part of the active site in staphylococcal nuclease. Transcription analysis revealed a specific 1.3-kb mRNA, which corresponded to the size predicted by the promoter and transcription termination signature sequences and indicated a monocistronic mode of transcription. Allelic replacement of the ORF rendered a M49 mutant devoid of extracellular DNase activity when cultured on indicator agar. Virulence parameters such as resistance to phagocytosis were not affected by the mutation. The sda gene was cloned and expressed in Escherichia coli as a thioredoxin fusion. By performing Ouchterlony immunodiffusion on the recombinant protein and by using protein preparations from culture supernatants of wild-type bacteria and the DNase mutant, the results of immunoreactivity with DNase type-specific polyclonal rabbit antisera classified the DNase as a type D enzyme. Fifty percent of patients with sera exhibiting high titers of antistreptolysin or anti-DNase B antibodies also had SdaD-reactive antibodies in comparison with <10% of serologically normal controls. While the value of recombinant SdaD for diagnostic purposes needs to be clarified, the isogenic DNase mutant pair of M49 should allow the significance of GAS DNase D as a bacterial virulence factor to be determined.

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5 alpha. Unlike other GAS-Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG3. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited > 95% homology to corresponding sections of other enn genes. The region of enn64/14 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The recombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152 bp open reading frame of emmL9 encoding the N-terminus was found to display > 90% homology to the corresponding section of enn64/14 but was < 50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG3. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have involved a set of flanking 7 bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.