Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

Abstract

dTo obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here. Streptococcus pyogenes is a Gram-positive bacterium that causes a range of diseases in humans, including pharyngitis, superficial and deep skin infections, acute rheumatic fever, poststreptococcal glomerulonephritis, bacteremia, and necrotizing fasciitis ("flesh-eating disease") (1, 2). Globally, there are over 700 million group A streptococcus (GAS) infections annually (3). It has been estimated that 9,000 to 11,500 cases of invasive GAS disease occur each year in the United States, resulting in 1,000 to 1,800 deaths annually (4; http://www.cdc.gov). Rheumatic fever, necrotizing fasciitis, and toxic shock syndrome are responsible for the high morbidity and mortality rates due to GAS infections (5).Advances in DNA sequencing and related technologies have facilitated genome-wide dissection of genetic events involved in colonization (6), immune evasion (7), virulence (8-10), and the evolution and spread of highly virulent S. pyogenes clones (11-13). To advance our understanding of S. pyogenes molecular-pathogenesis events occurring during invasive infection, we performed whole-genome sequencing of 2,954 isolates recovered from a nonhuman primate model of necrotizing fasciitis. The resulting genome sequence data stimulated us to construct an isogenic fabT deletion mutant strain and to compare its global transcriptome and virulence attributes with those of the wild-type parental strain. MATERIALS AND METHODSBacterial strains and growth conditions. Escherichia coli strai...

The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

“…Recent studies have linked FabT-mediated regulation of fatty acid synthesis in GAS to virulence and intrahost genetic variation in invasive disease (55). In the ()cpsY mutant strain, the majority of the fabKDGFZ-accABCD operon was repressed (Table 1), as was the enoyl coenzyme A (enoyl-CoA) hydratase-encoding phaB.…”

Genetic heterogeneity of the Spy1336/R28 – Spy1337 Virulence Axis inStreptococcus pyogenesand Effect on Gene Transcript Levels and Pathogenesis

“…Primer sets 1336-1 and -2 and 1336-3 and -4 were used to amplify two fragments upstream and downstream, respectively, of Spy1336/R28. isogenic strains was performed as described previously (38,96,98). Bioanalyzer.…”

“…Briefly, mice were inoculated in the right hindlimb with 5x10 8 CFU (n=40 mice per strain) and followed for 7 days. A well-described NHP model of necrotizing myositis was used to compare the virulence of the wildtype and isogenic MGAS27961-10T-ΔSpy1336/R28 and MGAS27961-10T-ΔSpy1337 strains (96,99). Five cynomolgus macaques (2-3 years, 2-4 kg) were used.…”

“…R28 repeat region. Chromosomal DNA extraction was performed as described(96), using Fast-Prep lysing Matrix B beads in 2-ml tubes (M P Biomedicals), or the DNeasy blood and tissue kit (Qiagen). The primer sequences used for PCR-based size determination of the Spy1336/R28 repeat region are shown inTable S5.…”

Gene fitness landscape of group A streptococcus during necrotizing myositis

“…Analysis of the molecular pathogenesis of GAS necrotizing myositis requires use of appropriate animal models. Toward this end, mouse and nonhuman primate (NHP) necrotizing myositis models have been developed that approximate this disease (10, 33, 34). Importantly, GAS is a human-specific pathogen.…”