Gene fitness landscape of group A streptococcus during necrotizing myositis

Abstract

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...

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

“…60,100e102 Consistent with the data reported herein, transposon mutagenesis studies have shown rocA inactivation to significantly increase GAS fitness in mouse s.c. and NHP necrotizing myositis infection models. 60,101 The RNA-seq data demonstrate that rocA polymorphisms and rocA gene deletion significantly alter expression of many proven and putative virulence factors (Figure 3 and Supplemental Table S6). 43 As a result, rocA polymorphisms significantly increase M28 GAS virulence ( Figure 5).…”

“…Deletion of a putative methionine transporter system (encoded by M28_Spy0263/0264/0265) and two putative export systems (encoded by M28_Spy0625/0626/0627 and M28_Spy1711/1712) results in decreased fitness in human saliva ex vivo and NHP muscle in vivo. 60,102 In addition, genes encoding several proven virulence factors regulated by RocA also contribute to GAS fitness in various anatomic niches ( Supplemental Table S8). 23,25,60,65,66,100e108 Furthermore, increased expression of nga and slo ( Figure 3 and Supplemental Table S8) was recently implicated in the emergence and global dissemination of epidemic serotype M1 and M89 GAS strains.…”

“…111,112 Interestingly, the transposon mutagenesis library screens found deletion of fasBCA to result in increased GAS fitness for serotype M28, but not M1, in NHP necrotizing myositis. 60 Thus, the RocA and FasBCA/fasX systems may have serotype-specific regulatory functions.…”

“…A well-described NHP model of necrotizing fasciitis/ myositis was used. 52,58,60 Briefly, NHPs (n Z 8) were sedated and inoculated at a uniform depth with 1 Â 10 9 colony-forming units/kg in the left or right quadriceps muscle (n Z 4 limbs per strain). Animals were observed and necropsied 24 hours after infection.…”

Fitness genes of group A streptococci in necrotizing fasciitis and myositis

“…Although genome-wide transposon mutagenesis has been employed to investigate GAS fitness genes ex vivo in saliva and blood samples, there are no data from samples from human or nonhuman primates (NHP) with myositis. Since group A streptococcal infections are relatively specific in their presentation in humans and NHP compared with other animal species, such as mice, Zhu and colleagues (14) took the excellent approach of collaborating with the Animal Health Trust (Sufffolk, United Kingdom) as well as with the Department of Veterinary Medicine, University of Cambridge (Cambridge, United Kingdom) to carry out these critical studies in the NHP cynomolgus macaques, which are similar to humans (Figure 1). Six animals were inoculated intramuscularly with the GAS strains M1 or M28, followed by necropsy and muscle biopsy after 28 hours.…”