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...

Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes.

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.

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.