Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection

Breton, Yoann Le; Belew, Ashton T.; Freiberg, Jeffrey A.; Sundar, Ganesh S.; Islam, Emrul; Lieberman, Joshua A.; Shirtliff, Mark E.; Tettelin, Hervé; El-Sayed, Najib M.; McIver, Kevin S.

doi:10.1371/journal.ppat.1006584

Cited by 47 publications

(81 citation statements)

References 92 publications

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“…To validate the Tn-seq findings, we confirmed the involvement of three genes (dltA, lytR, and gacI) by comparing hGIIA-mediated killing of WT GAS with previously generated GAS mutants [32,42,44]. Deletion of dltA and lytR indeed increased GAS susceptibility to hGIIAmediated killing by 45-fold and 35-fold, respectively (Fig 2A, B).…”

Section: Hgiia Requires the Gac Glcnac Side Chain To Exert Its Bactersupporting

confidence: 56%

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Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Hensbergen

Movert

Maat

et al. 2018

Preprint

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23Human Group IIA secreted phospholipase A 2 (hGIIA) is an acute phase protein with 24 bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic 25 mice have highlighted the importance of hGIIA as an innate defense mechanism against the 26 human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). 27Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To 28 identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon 29 library to recombinant human hGIIA and compared relative mutant abundance with library 30 input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the 31 output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA 32 susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included 33 two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene 34 cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented 35 strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in 36 the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is 37 conserved across different GAS serotypes. Increased resistance is associated with delayed 38 penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B 39Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. 40This suggests that the streptococcal Lancefield antigens, which are critical determinants for 41 streptococcal physiology and virulence, are required for the human bactericidal enzyme 42 hGIIA to exert its bactericidal function.

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Section: Hgiia Requires the Gac Glcnac Side Chain To Exert Its Bactersupporting

confidence: 56%

“…The GAS M1T1 5448 strain was used in this study unless stated otherwise. The 5448ΔgacI knockout and gacI* complemented strain [32], the 5448ΔlytR [44] and the GAS serotypes M2, M3, M4, and M28 and corresponding Δ gacI knockouts [74] were described previously.…”

Section: Bacterial Strains and Serummentioning

confidence: 99%

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Hensbergen

Movert

Maat

et al. 2018

Preprint

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“…Tn-seq Krmit transposon insertion tags were prepared from the cell pellets as previously described 15,87 13 C, 31 P 37 spectra were obtained using echo/antiecho gradient selection and constant time in t 2 with a nominal value of n J CP of 5 Hz and without multiplicity selection. The spectra were processed and analyzed using TopSpin 4.0.1 software (Bruker BioSpin).…”

Section: Tn-seq Library Screen For Zn 2+ Sensitivitymentioning

confidence: 99%

“…Tn-seq read datasets were analyzed (quality, filtering, trimming, alignment, visualization) as previously described15,87 using the M1T1 5448 genome as reference for read alignments. The ratios of mutant abundance comparing the output to input mutant pools were calculated as a fold change for each GAS gene using the DEseq2 and EdgeR pipelines[87][88][89] . Illumina sequencing reads from the Tn-seq analysis were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP150081.…”

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confidence: 99%

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

Edgar

Hensbergen

Ruda

et al. 2018

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Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. These structures have also gained interest as vaccine antigens, in particular for the human pathogens Group A Streptococcus (GAS) and Streptococcus mutans. Streptococcal cell wall glycopolymers are considered to be functional homologues of wall teichoic acids but surprisingly lack the biologically-relevant and characteristic anionic charge. Here we identify gacH, a gene of unknown function in the GAS Group A Carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA secreted phospholipase A2. To understand the underlying mechanism of these phenotypes, we determined the structure of the extracellular domain of GacH and discover that it represents a new family of glycerol phosphate (GroP) transferases. Importantly, we demonstrate the presence of GroP in both the GAC and the homologous Serotype c Carbohydrate (SCC) from S. mutans, which is conferred by gacH and sccH products, respectively. NMR analysis of GAC released from cell wall by non-destructive methods reveals that approximately 30% of the GAC GlcNAc side-chains are modified by GroP at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.Graphical abstract

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“…Technologies such as signature-tagged mutagenesis (STM), transposon site hybridization (TraSH), and Tn-seq have been applied successfully to many bacterial pathogens to identify genes required for fitness under diverse in vivo and ex vivo conditions (24–30). In GAS, genome-wide transposon mutagenesis screens have been used to identify genes contributing to fitness during growth in human blood ex vivo , human saliva ex vivo , and mouse subcutaneous infections (24, 30–32). However, a genome-wide investigation of the GAS genes contributing to fitness in necrotizing myositis has not been undertaken.…”

Section: Introductionmentioning

confidence: 99%

Gene fitness landscape of group A streptococcus during necrotizing myositis

Zhu

Olsen

Beres

et al. 2018

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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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Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection

Cited by 47 publications

References 92 publications

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human group IIA secreted phospholipase A2 to exert its bactericidal effects

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides

Gene fitness landscape of group A streptococcus during necrotizing myositis

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