Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of
            <i>rocA</i>
            Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

Zhu, Luchang; Olsen, Randall J.; Horstmann, Nicola; Shelburne, Samuel A.; Fan, Jia; Hu, Ye; Musser, James M.

doi:10.1128/iai.00258-16

Cited by 21 publications

(36 citation statements)

References 40 publications

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“…MGAS2221 RocA-null mutants are more virulent than wild-type MGAS2221 in subcutaneous infections in mice, confirming the recent findings that RocA mutations or rocA-downregulating mutations enhance the virulence of GAS in different serotype backgrounds (24,(29)(30)(31). The rocA C605A mutant had higher levels of expression of hasA, spyCEP, scpaA, sda1, slo, nga, sagA, and sse than MGAS2221 in vivo, while the two strains had similar levels of speB expression in skin infections in mice.…”

Section: Discussionsupporting

confidence: 75%

“…RocA mutations are detected in clinical M1 GAS isolates (31). Mutations that lead to the loss of RocA expression or function enhance virulence (24,30,31). Whether null CovS and RocA mutations have any distinct effects on gene expression is not known.…”

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confidence: 99%

“…In particular, all M3 GAS isolates isolated from diverse geographic locations since the 1930s have a truncation mutation that enhances the levels of the hyaluronic acid capsule (28), and a nonsense mutation in rocA is present in serotype M18 GAS isolates and contributes to the hyperencapsulation of serotype M18 strains (29). Serotype M89 strains have polymorphisms in the region upstream of the rocA gene that alter rocA expression (30). RocA mutations are detected in clinical M1 GAS isolates (31).…”

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confidence: 99%

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Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

et al. 2017

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Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse Aϩ ] and normal SpeB activity [SpeB Aϩ ]). Sse Aϩ SpeB Aϩ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse Aϩ SpeB Aϩ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse Aϩ SpeB Aϩ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.KEYWORDS CovRS, PAF acetylhydrolase, RocA, SpeB, Streptococcus pyogenes, group A Streptococcus, in vivo expression, mutation, virulence, virulence regulation T he human pathogen group A Streptococcus (GAS) commonly causes pharyngitis and superficial skin infections. GAS also causes severe invasive infections, such as necrotizing fasciitis and sepsis. The most recently available data indicate that from 2005 to 2012 severe invasive infections in the United States were most frequently associated with GAS of the M protein serotypes M1, M12, M28, M89, and M3 (1), and the currently circulating M1 GAS belongs to the pandemic M1T1 clone (2). GAS produces an

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Section: Discussionsupporting

confidence: 75%

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confidence: 99%

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confidence: 99%

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Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

et al. 2017

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“…While serotype M3 and M18 GAS are the only known serotypes that are exclusively rocA mutant, individual isolates of other serotypes have also been identified as rocA mutant (22-25, 44, 47, 48). Data from several studies are consistent with rocA mutant strains being selected for from rocA ϩ parental isolates, and that selection primarily occurs during invasive GAS infection (44,(47)(48)(49). This mirrors the more than 15 years worth of data concerning the selection of covR or covS mutant strains during invasive GAS infections (27)(28)(29)(30)(31)(32)(33)(34)50).…”

Section: Discussionsupporting

confidence: 55%

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus

et al. 2017

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Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg 2ϩ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.

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“…That is, inactivating these genes potentially confers enhanced GAS fitness during NHP necrotizing myositis. These genes include known negative regulators of virulence rivR (60) and rocA (61–65). Of note, rocA and ppiB (peptidyl-prolyl cis-trans isomerase) were identified in both the serotype M1 and M28 strains (Figure 3B, Table S3, Table S5).…”

Section: Resultsmentioning

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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Intergenic Variable-Number Tandem-Repeat Polymorphism Upstream of rocA Alters Toxin Production and Enhances Virulence in Streptococcus pyogenes

Cited by 21 publications

References 40 publications

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus

Gene fitness landscape of group A streptococcus during necrotizing myositis

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