Attenuated expression of the mga virulence regulon in an M serotype 50 mouse-virulent group A streptococcal strain.

The attenuated expression of virulence genes found in a group A streptococcal strain that is naturally pathogenic for mice was postulated to result from a defect in the strain's multigene regulator, Mga. The sequence of the mga gene reveals three amino acid changes in the gene product that might affect protein function. The defect in the mga gene was complemented by providing either the closely similar mga4 allele or a more divergent mga1 allele in trans. Complementation increased the amount of emm50 transcript and the quantity of surface-extractable M protein, restoring virulence function.

Role of putative virulence factors of Streptococcus pyogenes in mouse models of long-term throat colonization and pneumonia.

To investigate the role of putative virulence factors of Streptococcus pyogenes (group A streptococcus; GAS) in causing disease, we introduced specific mutations in GAS strain B514, a natural mouse pathogen, and tested the mutant strains in two models of infection. To study late stages of disease, we used our previously described mouse model (C3HeB/FeJ mice) in which pneumonia and systemic spread of the streptococcus follow intratracheal inoculation. To study the early stages of disease, we report here a model of long-term (at least 21 days) throat colonization following intranasal inoculation of C57BL/10SnJ mice. When the three emm family genes of GAS strain B514-Sm were deleted, the mutant showed no significant difference from the wild type in induction of long-term throat colonization or pneumonia. We inactivated the scpA gene, which encodes a complement C5a peptidase, by insertion of a nonreplicative plasmid and found no significant difference from the wild type in the incidence of throat colonization. However, there was a small but statistically significant decrease in the incidence of pneumonia caused by the scpA mutant. Finally, we demonstrated a very important effect of the hyaluronic acid capsule in both models. Following intranasal inoculation of mice with a mutant in which a nonreplicative plasmid was inserted into the hasA gene, which encodes hyaluronate synthase, we found that all bacteria recovered from the throats of the mice were encapsulated revertants. Following intratracheal inoculation with the hasA mutant, the incidence of pneumonia within 72 h was significantly reduced from that of the control strain (P = 0.006). These results indicate that the hyaluronic acid capsule of S. pyogenes B514 confers an important selective advantage for survival of the bacteria in the upper respiratory tract and is also an important determinant in induction of pneumonia in our model system.

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 natural 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 colonization and disease. The emm gene cluster for this strain was examined by PCR mapping and found to contain three emm family genes and cluster pattern 5. PCR-generated fragments corresponding to the SF4 (mrp50), SF2 (emmL50), and SF3 (enn50) genes were cloned and the entire gene cluster was sequenced. The gene cluster has greater than 97% DNA identity to previously sequenced regions of the gene cluster of the M2 strain T2/44/RB4 if two small divergent regions that encode the mature amino terminus of the SF-2 and SF-3 gene products are not included. If expressed, the genes encode proteins which bind human immunoglobulin G (Mrp50 and EmmL50) or immunoglobulin A (Enn50). However, in isolates taken directly after passage in mice, the surface proteins arising from these genes were barely detectable. The transcription of each gene in the B514 strain was investigated by Northern (RNA) hybridization, and mRNA transcripts were detected and quantitated relative to those of the recA gene, a housekeeping gene. Transcription of all three emm family genes was found to be over 30-fold attenuated relative to transcription of 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 different requirement for activation; it also suggests that the capsule may be sufficient to inhibit phagocytosis under these circumstances.

Structural heterogeneity of the emm gene cluster in group A streptococci.

One or more distinct copies of emm genes lie within a gene cluster that is located downstream from a transcriptional regulatory gene (mry). Mry is a positive regulator for the genes in this cluster and for the downstream gene, scpA. The objective of this study is to examine the structure of this cluster and the distribution of specific alleles within the cluster among group A streptococcal isolates of 32 different serotypes. The peptidoglycan (PG)-spanning domain, which exists in four divergent forms, was used to identify specific alleles of the genes within the emm cluster. Gene content of the cluster was determined by Southern hybridization with allele-specific oligonucleotides. Five different chromosomal patterns for this cluster were observed. Sequence heterogeneity in the adjacent mry locus was demonstrated by the ability of some of the isolates to hybridize with a whole mry gene probe, but not with mry-based oligonucleotide probes. A PCR-based chromosomal mapping technique was used to examine further the gene order within the emm gene clusters. Structural heterogeneity of the emm gene cluster was found within class I isolates in this study, while class II isolates were relatively homogeneous at this chromosomal locus and distinct from class I.