DNA-SEQUENCING AND GENE-EXPRESSION OF THE EMM GENE-CLUSTER IN AN M50 GROUP-A STREPTOCOCCUS STRAIN VIRULENT FOR MICE

The strain B514, an M serotype 50 strain, is capable of causing a natu ral upper respiratory infection leading to death in mice, as reported by Hook et al. in 1960 (E. W. Hook, R. R. Wagner, and R. C. Lancefield , Am. J. Hyg, 72:111-119, 1960). Thus, this strain was of interest for use in developing an animal model for group A streptococcal colonizat ion and disease. The emm gene cluster for this strain was examined by PCR mapping and found to contain three emm family genes and cluster pa ttern 5. PCR-generated fragments corresponding to the SF4 (mrp50), SF2 (emmL50), and SF3 (enn50) genes were cloned and the entire gene clust er was sequenced. The gene cluster has greater than 97% DNA identity t o previously sequenced regions of the gene cluster of the M2 strain T2 /44/RB4 if two small divergent regions that encode the mature amino te rminus of the SF-2 and SF-3 gene products are not included. If express ed, the genes encode proteins which bind human immunoglobulin G (Mrp50 and EmmL50) or immunoglobulin A (Enn50). However, in isolates taken d irectly after passage in mice, the surface proteins arising from these genes were barely detectable. The transcription of each gene in the B 514 strain was investigated by Northern (RNA) hybridization, and mRNA transcripts were detected and quantitated relative to those of the rec A gene, a housekeeping gene. Transcription of all three emm family gen es was found to be over 30-fold attenuated relative to transcription o f the same genes in strain T2/44/ RB4. This suggests that the positive regulator, Mga, either is not expressed in this strain or has a diffe rent requirement for activation; it also suggests that the capsule may be sufficient to inhibit phagocytosis under these circumstances.