We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD + -glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.pathogenesis | phylogeography | mobile genetic element | flesh-eating disease | molecular clock

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...

Faecalibacterium prausnitzii is considered as one of the most important bacterial indicators 32 of a healthy gut. We studied the effects of oral F. prausnitzii treatment on high-fat fed mice. 33Compared to the High-fat Control mice, F. prausnitzii-treated mice had lower hepatic fat 34 content, AST and ALT, and increased fatty-acid oxidation and adiponectin signaling in liver.

The sequences reported in this paper have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (see URLs) with the Bioproject accession number PRJNA434389 and the NCBI Gene Expression Omnibus (GEO) under accession number GSE113058. ETHICS STATEMENTAll mouse studies were performed in accordance with a protocol (AUP-0318-0016) approved by the Institutional Animal Care and Use Committee at Houston Methodist Research Institute. All studies with human blood and blood components were performed in accordance with a protocol (01-I-N055) approved by the Institutional Review Board for human subjects, National Institute of Allergy and Infectious Diseases. All study volunteers gave written informed consent.