R e s e a R c h a R t i c l e 3 5 4 6jci.org Volume 125 Number 9 September 2015 ogous recombinational molecular genetic event resulted in upregulation of production of extracellular NADase and SLO toxins and recently stimulated an epidemic of serotype M89 GAS infections caused by a recently emerged clone.
ResultsPre-epidemic and epidemic serotype M1 strains. Mouse invasive infection and nonhuman primate pharyngitis and invasive infection studies show that the MGAS2221 strain, genetically representative of the epidemic ("new") clone, is more virulent than reference strain SF370, genetically representative of pre-epidemic ("old") serotype M1 strains (3). Strain MGAS2221 lacks polymorphisms in regulatory genes known to influence virulence, such as covRS, ropB, and mga. A previous expression microarray analysis of the transcriptomes of pre-epidemic and epidemic M1 strains found that only 8 core chromosomal genes were differentially transcribed between the two strain groups in early logarithmic growth phase (12). Given the clear evidence that pre-epidemic and epidemic M1 strains differ significantly in virulence, we tested the hypothesis that the expression microarray analysis had failed to identify critical virulence genes differentially regulated between the two strain groups. We reassessed global transcriptome differences in a panel of pre-epidemic and epidemic M1 strains using RNA sequencing (RNA-Seq) analysis, a more sensitive and accurate method of transcript analysis. RNA-Seq analysis detected 5 differentially expressed genes, confirming our previous expression microarray findings of very limited gene transcript differences between pre-epidemic and epidemic strains. Of particular interest, all 5 differentially regulated genes were located in the 36-kb region of recombinational replacement. These genes include nga, ifs (encoding Ifs, an endogenous inhibitor of SPN; refs. 15, 16), slo, and two small genes of uncertain function (Spy0142 and Spy0144; SPN enhances GAS survival by inhibiting pathogen internalization by host cells and also augments SLO cytotoxicity (7-10). SLO is a potent oxygen-sensitive cytolytic toxin that forms pores in hostcell membranes (10). The coordinated activities of SPN and SLO prevent maturation of phagolysosomes and thereby decrease phagocytic killing of GAS (9, 11). Several lines of evidence suggest that the nga and slo genes may play a critical role in M1 epidemicity. First, the transcript levels of nga and slo are significantly higher in epidemic strains than in preepidemic M1 GAS strains (12). Second, epidemic M1 strains produce more SPN and SLO activity than pre-epidemic strains (12,13). By comparing the genome sequences of a genetically representative epidemic (MGAS2221) strain and a pre-epidemic (SF370) strain, we discovered that the two strains differ by 59 SNPs and 2 indels in the nga-slo region of recombination (3). Among these SNPs, 3 were of particular interest because they are located in regions that, in principle, might influence nga and slo transcript level and strain phe...