Novel genes required for the fitness of<i>Streptococcus pyogenes</i>in human saliva

Zhu, Luchang; Charbonneau, Amelia R. L.; Waller, Andrew S.; Olsen, Randall J.; Beres, Stephen B.; Musser, James M.

doi:10.1101/196931

Cited by 8 publications

(22 citation statements)

References 63 publications

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“…The insertion index (number of unique insertions/size of the gene) of each of the genes in the M1 and M28 genomes is illustrated in Figure 1B. Use of the MGAS2221 transposon mutant library to identify novel genes contributing to GAS fitness in human saliva ex vivo has been described recently (31).…”

Section: Resultsmentioning

confidence: 99%

“…That is, we were able to assess the extent to which GAS has infection-specific genetic programs. We recently reported that 92 serotype M1 genes were required for optimal growth ex vivo in human saliva (31). Only 19 (21% of 92) genes were defined as contributing to serotype M1 fitness in both human saliva and NHP necrotizing myositis (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

“…Here we report the first use in NHPs of a relatively new genome-wide transposon mutagenesis technique termed transposon directed insertion-site sequencing (TraDIS) (41, 42). TraDIS was recently documented to provide much novel information about GAS genes contributing to fitness in human saliva (31). Using highly saturated transposon mutant libraries made in two genetically distinct GAS strains that are common causes of severe human infections, we identified novel genes required for bacterial fitness during necrotizing myositis.…”

Section: Introductionmentioning

confidence: 99%

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Gene fitness landscape of group A streptococcus during necrotizing myositis

Zhu

Olsen

Beres

et al. 2018

Preprint

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23[ABSTRACT] 24Necrotizing fasciitis and myositis are devastating infections characterized 25 by high mortality. Group A streptococcus (GAS) is a common cause of 26 these infections, but the molecular pathogenesis is poorly understood. We 27 report a genome-wide analysis using serotype M1 and M28 strains that 28 identified novel GAS genes contributing to necrotizing myositis in 29 nonhuman primates (NHP), a clinically relevant model. Using transposon 30 directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS 31 genes required for infection by serotype M1 and M28 organisms, 32 respectively. For both M1 and M28 strains, more than 25% of the GAS 33 genes required for necrotizing myositis encode known or putative 34 transporters. Thirteen GAS transporters contributed to both M1 and M28 35 strain fitness in NHP myositis, including putative importers for amino 36 acids, carbohydrates, and vitamins, and exporters for toxins, quorum 37 sensing peptides, and uncharacterized molecules. Targeted deletion of 38 genes encoding five transporters confirmed that each isogenic mutant 39 strain was significantly impaired in causing necrotizing myositis in NHPs. 40 qRT-PCR analysis showed that these five genes are expressed in infected 41 NHP and human skeletal muscle. Certain substrate-binding lipoproteins of 42 these transporters, such as Spy0271 and Spy1728, were previously 43 documented to be surface-exposed, suggesting that our findings have 44 translational research implications. 45 46 revealed some of the GAS molecules that contribute to the pathogenesis of 63 necrotizing fasciitis and myositis, including M protein (8, 9), extracellular cysteine 64 protease streptococcal pyrogenic exotoxin B (SpeB) (10-13), hyaluronic acid 65 capsule (14), and cytotoxins NADase and streptolysin O (15-21). However, 66although the genome of GAS is relatively small (~1,800 genes) (22, 23), current 67 understanding of the molecular pathogenesis of GAS necrotizing fasciitis and 68 myositis is limited. 69High-throughput genome-wide screens based on transposon mutagenesis 70 strategies are very useful in providing new information about the genetic basis of 71 bacterial virulence. Technologies such as signature-tagged mutagenesis (STM), 72 transposon site hybridization (TraSH), and Tn-seq have been applied 73 successfully to many bacterial pathogens to identify genes required for fitness 74 under diverse in vivo and ex vivo conditions (24-30). In GAS, genome-wide 75 transposon mutagenesis screens have been used to identify genes contributing 76 to fitness during growth in human blood ex vivo, human saliva ex vivo, and 77 mouse subcutaneous infections (24, 30-32). However, a genome-wide 78 investigation of the GAS genes contributing to fitness in necrotizing myositis has 79 not been undertaken. 80Analysis of the molecular pathogenesis of GAS necrotizing myositis 81 requires use of appropriate animal models. Toward this end, mouse and 82 nonhuman primate (NHP) necrotizing myositis models have been developed that 83 approximate this d...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gene fitness landscape of group A streptococcus during necrotizing myositis

Zhu

Olsen

Beres

et al. 2018

Preprint

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“…Most genes directly or indirectly involved in the GAC biosynthesis pathway are essential for GAS viability, including all four dTDP-L-rhamnose biosynthesis genes rmlABC and gacA (Le Breton et al, 2013;van der Beek et al, 2015;Zhu et al, 2017). In contrast and for unknown reasons, the dTDP-L-rhamnose biosynthesis genes are not essential in S. mutans under noncompetitive conditions.…”

Section: Gas Rmlb and Rmlc Functionally Replace S Mutans Homologsmentioning

confidence: 99%

“…In contrast, all GAS serotypes express a structurally invariant GAC (Lancefield, 1933). Similar to classical wall teichoic acids, rhamnose cell wall polysaccharides are critical for maintaining cell shape, bacterial physiology and virulence, but in-depth knowledge of their biosynthesis or host interactions at a molecular level is limited (van Sorge et al, 2014;van der Beek et al, 2015;Kovacs et al, 2017;Zhu et al, 2017). A better understanding of these mechanisms could aid the development of new classes of antibiotics, antibiotic adjuvants or vaccines (Sabharwal et al, 2006;van Sorge et al, 2014;St Michael et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Beek

Zorzoli

Çanak

et al. 2019

Molecular Microbiology

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Summary Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus ; GAS), Streptococcus mutans and Mycobacterium tuberculosis . Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans , we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC 50 of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.

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Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus

Bernard

Kachroo

Eraso

et al. 2019

The American Journal of Pathology

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Two-component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine-tune their activity. In group A Streptococcus (GAS), regulator of Cov (RocA) is an accessory protein that functions with the control of virulence regulator/sensor TCS, which regulates approximately 15% of the GAS transcriptome. Whole-genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acidealtering) and nonsense (protein-truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally occurring clinical isolates with rocA polymorphisms (n Z 48), an isogenic rocA deletion mutant strain, and five isogenic rocA polymorphism mutant strains to perform genome-wide transcript analysis (RNA sequencing), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change, causing a wild-typeelike virulence phenotype, or a substantial transcriptome change, leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Taken together, our data show that naturally occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.

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Novel genes required for the fitness ofStreptococcus pyogenesin human saliva

Abstract: Streptococcus pyogenes (group A Streptococcus, or GAS) causes 23 600 million cases of pharyngitis each year. Despite this considerable disease 24

Cited by 8 publications

References 63 publications

Gene fitness landscape of group A streptococcus during necrotizing myositis

Gene fitness landscape of group A streptococcus during necrotizing myositis

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus

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