ABSTRACT
Necrotizing fasciitis and myositis are devastating infections characterized by high mortality. Group A streptococcus (GAS) is a common cause of these infections, but the molecular pathogenesis is poorly understood. We report a genome-wide analysis using serotype M1 and M28 strains that identified novel GAS genes contributing to necrotizing myositis in nonhuman primates (NHP), a clinically relevant model. Using transposon directed insertion-site sequencing (TraDIS) we identified 126 and 116 GAS genes required for infection by serotype M1 and M28 organisms, respectively. For both M1 and M28 strains, more than 25% of the GAS genes required for necrotizing myositis encode known or putative transporters. Thirteen GAS transporters contributed to both M1 and M28 strain fitness in NHP myositis, including putative importers for amino acids, carbohydrates, and vitamins, and exporters for toxins, quorum sensing peptides, and uncharacterized molecules. Targeted deletion of genes encoding five transporters confirmed that each isogenic mutant strain was significantly impaired in causing necrotizing myositis in NHPs. qRT-PCR analysis showed that these five genes are expressed in infected NHP and human skeletal muscle. Certain substrate-binding lipoproteins of these transporters, such as Spy0271 and Spy1728, were previously documented to be surface-exposed, suggesting that our findings have translational research implications.
Introduction
Necrotizing fasciitis, commonly known as the “flesh-eating disease”, is an invasive infection with very high rates of human morbidity and mortality (1, 2). In severe cases, contiguous muscle may be severely damaged, resulting in necrotizing myositis. Based on whether the cause is polymicrobial or monomicrobial, necrotizing fasciitis can be divided into type I (polymicrobial) and type II (monomicrobial) (2, 3). Group A streptococcus (GAS) is the primary cause of type II necrotizing fasciitis, and has an average case fatality rate of 29% (2, 4, 5). The molecular pathogenesis processes at work in GAS necrotizing fasciitis and myositis are poorly understood, a lack of knowledge that has impeded development of new effective diagnostics and therapeutics.
GAS is a human-specific pathogen causing more than 700 million infections annually worldwide (6). GAS infections range from relatively benign pharyngitis, skin, and soft-tissue infections, to life-threatening invasive diseases such as necrotizing fasciitis and necrotizing myositis (1, 7). No vaccine is currently available to prevent GAS infections. Decades of research have revealed some of the GAS molecules that contribute to the pathogenesis of necrotizing fasciitis and myositis, including M protein (8, 9), extracellular cysteine protease streptococcal pyrogenic exotoxin B (SpeB) (10–13), hyaluronic acid capsule (14), and cytotoxins NADase and streptolysin O (15–21). However, although the genome of GAS is relatively small (~1,800 genes) (22, 23), current understanding of the molecular pathogenesis of GAS necrotizing fasciitis and myositis is limited.
High-throughput genome-wide screens based on transposon mutagenesis strategies are very useful in providing new information about the genetic basis of bacterial virulence. 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.
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. Some GAS virulence factors are specific for human and NHP target molecules (35–38) and have significantly decreased or no activity against the mouse homologs (38–40). Thus, NHP necrotizing myositis provides the most relevant experimental model possible.
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. Our screen revealed the theme that GAS transporters play a pivotal role in this infection. We confirmed and extended the TraDIS screen data using isogenic mutant strains, in vitro growth phenotyping and qRT-PCR analysis of necrotic myositis tissues taken from infected NHPs and human patients.
Results
Construction of highly saturated transposon mutant libraries in genetically representative strains of serotype M1 and M28 of GAS
Transposon insertion mutant libraries were generated using serotype M1 strain MGAS2221 and serotype M28 strain MGAS27961 as the parental organisms. Strains of these two M protein serotypes were used because they are among the five more abundant M protein types causing invasive infections in many countries, and in some cases they are the dominant causes of infections. Thus, serotype M1 and M28 GAS are clinically highly relevant (43–46). These two strains were chosen for transposon mutagenesis because (i) strain MGAS2221 is genetically representative of a pandemic serotype M1 clone that arose in the 1980s, rapidly spread worldwide and currently is the most prevalent cause of severe infections globally (18, 20, 47), (ii) strain MGAS27961 is genetically representative of a virulent serotype M28 clone that is prevalent in the United States and elsewhere (48), (iii) both strains have wild-type alleles of all major transcriptional regulators that are known to affect GAS virulence, such as covR and covS, ropB, mga, and rocA, and (iv) both strains have been used previously in animal infection studies (18, 20, 33). Using transposon plasmid pGh9:ISS1 (49), we generated dense transposon mutant libraries in strains MGAS2221 and MGAS27961 containing 154,666 (an insertion every 12 bp on average) and 330,477 (an insertion every 5.5 bp on average) unique transposon insertions, respectively (Figure 1A). This means that on average, the serotype M1 and M28 libraries had 66 and 139 insertions per open reading frame. 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).
Genome-wide screens identify GAS genes contributing to fitness in a necrotizing myositis infection model in cynomolgus macaques
We screened the M1 and M28 GAS transposon mutant libraries in NHPs as a first step toward discovering genes contributing to fitness during necrotizing myositis. Six cynomolgus macaques each were inoculated by intramuscular injection with either the M1 or M28 transposon mutant libraries and followed for 24 h. All animals developed signs and symptoms consistent with necrotizing myositis and were necropsied. Biopsies containing necrotic muscle were obtained from the inoculation site to recover output mutant pools for subsequent analysis. Quantitative culture yielded an average of 4.87 × 108 CFU/g for M1, and 8.77 × 108 for M28 in the tissue biopsy specimens used for TraDIS analysis.
TraDIS was used to compare the mutant compositions of the input and output pools. The TraDIS analysis identified genes with significantly altered mutant frequency in the output mutant pools relative to the input mutant pool (examples shown in Figure S1). Infection bottlenecks can be a technical challenge for high-throughput transposon mutagenesis studies, and substantial loss of mutant library complexity during animal infection can result in erroneous identification of fitness genes (50). Our TraDIS results showed that for both M1 and M28 GAS screens, there was no substantial decline of mutant library complexity post NHP infections (Figure 2A,B). On average, 67% and 84% of the library complexity remain in the M1 and M28 output pools, respectively (Figure 2A,B). This high diversity of transposition site mutants recovered is inconsistent with a narrow infection bottleneck and indicates that our screens are unlikely to erroneously identify fitness genes. To identify GAS fitness genes in the infected NHP skeletal muscle milieu, genes previously identified as essential for GAS growth in vitro in rich medium (THY) were excluded from the analysis as is commonly done (51). Disrupted genes associated with significantly decreased fitness (transposon frequency log2 fold-change < −1, and q value < 0.01) in the output mutant pools were interpreted as contributing to NHP necrotizing myositis (Figure 2C,D, Figure 3). We identified 126 and 116 genes in the serotype M1 and M28 strains, respectively, that are crucial for GAS fitness in this infection model (Figure 3A). That is, inactivating these genes potentially confers diminished GAS fitness in necrotizing myositis. Importantly, a common set of 72 genes was identified as crucial for fitness in both the serotype M1 and M28 library NHP screens (Figure 3A, Table S1). The shared 72 genes represent 57% of the serotype M1 fitness genes and 65% of the serotype M28 fitness genes (Figure 3A). Functional categorization of the fitness genes found that numerically, the more prevalent GOG categories included genes inferred to be involved in amino acid transport and metabolism (E), inorganic ion transport and metabolism (P), and transcription (K) (Figure 3C). Genes encoding many documented virulence factors or virulence modulating factors were identified as contributing to fitness in both serotype M1 and M28 GAS strains, including adcB/C (52, 53), gacI (54, 55), pepO (56, 57), inlA (58), perR (59) and scfAB (32) (Table S1, Table S2, Table S4).
We also identified 21 and 20 genes in the serotype M1 and M28 strains, respectively, that are associated with significantly increased fitness in vivo (transposon insertion frequency log2 fold-change > 1, and q value < 0.01) (Figure 2, Figure 3B and 3D). 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). Inactivation of the rocA gene in an M28 GAS strain was recently shown to significantly increase virulence in a mouse model of necrotizing myositis (66).
To investigate the phylogenetic distribution of the identified in vivo fitness genes among diverse GAS strains, we examined the presence of M1 and M28 fitness genes required during NHP necrotizing myositis in 62 sequenced GAS genomes representing 26 different M protein serotypes (Table S6). The vast majority of the M1 fitness genes (96%) and M28 fitness genes (93%) are present in at least 61 of the 62 GAS strains (Figure 3, E and F).
Comparison of the genetic requirement for necrotizing myositis and those for GAS fitness in human saliva and blood ex vivo, and mouse subcutaneous infections
Previous genome-wide transposon mutagenesis studies identified GAS genes required for growth in animal infection models and ex vivo in human body fluids such as blood and saliva (30–32). These published data allowed us to test the hypothesis that the GAS gene requirements for NHP necrotizing myositis are distinct from those identified by transposon screens performed in other model infection environments. 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). These genes include metabolic genes (purA, purB, acoABCL, glgP and malM) and transporter genes (adcAB, braB, mtsA, mtsB, artP, and artQ). The great majority of M1 genes (n = 107, 85%) required in NHP necrotizing myositis did not overlap with fitness genes required ex vivo in human saliva. Using a similar transposon mutagenesis technique (Tn-seq) 147 genes were identified as contributing to fitness of serotype M1 GAS strain 5448 after subcutaneous inoculation in mice (32). The overlap between the mouse subcutaneous fitness genes and the 126 necrotizing myositis genes is relatively larger (n = 39) (Figure 4B). These genes include metabolic genes purA, purB, acoABCL, glgP, malM, arcABCD, and phosphotransferase system genes manMLN. However, 69% of the genes (n = 87) required for NHP necrotizing myositis did not overlap with the genes identified in the mouse subcutaneous infection study. Using a transposon mutagenesis technique, Mclver and colleagues (30) identified 81 M1 GAS genes required for optimal bacterial growth in human blood. In comparison with the 126 necrotizing myositis fitness genes, only 14 genes were required in both conditions (Figure 4C). 89% of the genes (n = 112) required for NHP necrotizing myositis did not overlap with the genes required in human blood ex vivo. These include genes for carbohydrate metabolism (glgP, malM), transporters (adcB, braB, mtsA, mtsB), and transcriptional regulators (adcR, ciaH, ciaR, ihk, irr). To summarize, there is only modest overlap between the GAS genes contributing to fitness during experimental NHP necrotizing myositis, relative to growth ex vivo in human saliva and blood, and mouse subcutaneous infection. These results are consistent with our hypothesis that GAS has infection-specific genetic programs.
Genes encoding transporters constitute a considerable portion of GAS fitness determinants in experimental NHP necrotizing myositis
Bioinformatic analyses of the identified fitness genes found that regardless of M1 or M28 serotype, more than 25% of the genes contributing to in vivo fitness during NHP necrotizing myositis encode proven or putative transporters (Figure 5A). Specifically, 25.4 % of the serotype M1 fitness genes (n = 32) and 29.7% of the M28 GAS fitness genes (n = 32) encode proven or putative transporters (Figure 5A). Importantly, 26 transporter genes are required in both M1 and M28 strains during infection, indicating that there was considerable overlap between the two sets of genes (Figure 5B). These 26 shared genes encode 13 distinct transporters with proven or predicted roles in uptake of nutrients such as amino acids, metal ions, vitamins, carbohydrate, and export of a variety of substrates. (Figure 5C). The DNA sequences of the 26 transporter genes are highly conserved (95% to 100% identity) among genomes for 62 sequenced GAS strains representing 26 different M protein serotypes (Figure 6). One gene (Spy0499) has less homology in GAS (81% identical in four GAS strains) among the 62 strains with complete genomes.
Validation of the TraDIS screen results for genes encoding putative amino acid transporters
Six of the 13 transporters identified to be important for both serotype M1 and M28 strains during NHP necrotizing myositis are putative amino acid transporters (Figure 5C, yellow). For example, Spy0014 is a putative amino acid permease and BraB is a putative branched-chain amino acid transporter. Spy0271, Spy0272, and Spy0273 constitute a putative ABC transporter with similarity to methionine transporter proteins MetQ (65% identical), MetN (73% identical), and MetP (71% identical), respectively, of Streptococcus pneumoniae (67).
To test the hypothesis that Spy0014, BraB, and Spy0271-0273 participate in amino acid transport, we constructed isogenic deletion mutant strains ΔSpy0014, ΔbraB, and ΔSpy0271-0273 in parental M1 strain MGAS2221. We studied their growth phenotypes in rich medium (THY broth), and in a peptide-free chemically defined medium (CDM) (Figure 7). Compared to the wild-type parental strain, the three isogenic mutant strains do not have a growth defect in THY medium (Figure 7A). However, the mutant strains had a severe growth defect when cultured in CDM (Figure 7B), a result consistent with our hypothesis. As anticipated, the growth defect of these three isogenic mutant strains ΔSpy0014, ΔbraB, and ΔSpy0271-0273 was ameliorated by supplementing CDM with 0.1 g/ml tryptone, a source of abundant peptides (Figure 7C). Together, these results are consistent with the idea that Spy0014, BraB, and Spy0271-0273 are amino acid transporters that are essential for GAS growth in the absence of a source of abundant exogenous peptides.
GAS is auxotrophic for 15 amino acids considered essential for growth (68, 69). We hypothesized that transporters Spy0014, BraB and Spy0271-0273 contribute to uptake of specific essential amino acids. To test this hypothesis, we supplemented CDM with a high concentration (1 g/L) of each of the highly soluble essential amino acids to determine if certain amino acids restored the growth of these transporter mutants via non-specific uptake (Figure 7D-F). Consistent with the hypothesis, supplementing CDM with methionine restored the growth of mutant ΔSpy0271-0273 to near-wild-type growth phenotype, suggesting that Spy0271-0273 encode a methionine transporter (Figure 7E). Similarly, supplementing CDM with histidine and valine partially restored the growth of mutant strains ΔSpy0014 and ΔbraB, respectively, suggesting that Spy0014 and BraB contribute to uptake of histidine and valine (Figure 7, D and F).
Validation of the TraDIS screen results in the NHP model of necrotizing myositis
To validate the TraDIS screen results in vivo, we infected NHPs in the quadriceps with parental M1 strain MGAS2221, and isogenic mutant strains ΔSpy0014, ΔbraB, and ΔSpy0271-0273. Compared to the wild-type parental strain, each of these three transporter mutant strains caused significantly smaller lesions characterized by less tissue destruction in the NHP necrotizing myositis model (Figure 8, A and B). In addition, compared to the wild-type parental strain, significantly fewer CFUs of each isogenic mutant strain were recovered from the inoculation site and a distal site of dissemination (Figure 8, C and D).
Virulence role of Spy1726-1728, a poorly characterized ABC transporter
Our genome-wide screens identified a putative ABC transporter of unknown function that is required for NHP infection by the M1 and M28 GAS strains (Figure 5C, red). This putative transporter is encoded by three contiguous genes: Spy1726 (transporter permease protein), Spy1727 (ATP-binding protein), and Spy1728 (substrate-binding lipoprotein). To confirm the virulence role of this putative transporter, we used isogenic mutant strain ΔSpy1726-1728 made by deleting the entire Spy1726-1728 region in serotype M1 parental strain MGAS2221. Consistent with the result from the initial NHP necrotizing myositis TraDIS screen, isogenic mutant strain ΔSpy1726-1728 is significantly attenuated in capacity to cause necrotizing myositis in NHPs (Figure 8, A - D). This putative ABC transporter operon was not identified as important for virulence in previous GAS transposon mutagenesis screens (24, 30, 32), suggesting an infection- or primate-specific role in necrotizing myositis. Of note, Spy1728 (a substrate-binding lipoprotein) was previously shown to be a GAS surface protein and potential vaccine candidate (70).
Virulence role of quorum sensing peptide transporter PptAB
Our genome-wide TraDIS screens suggest that inactivating the quorum sensing peptide transporter PptAB in the serotype M1 and M28 strains results in significantly decreased fitness in NHP necrotizing myositis. To test this finding, we generated isogenic mutant strain ΔpptAB by deleting the pptAB genes in serotype M1 parental strain MGAS2221. Relative to the WT parental strain, the ΔpptAB mutant strain is significantly attenuated in ability to cause necrotizing myositis in NHPs (Figure 8, A-D). PptAB has been reported to be required for exporting the SHP2 and SHP3 quorum sensing peptides (71). However, Rgg2, the transcriptional regulator that controls expression of the shp2 and shp3 genes was not identified as important for NHP infections in our TraDIS screens. These results suggest the attenuation of the virulence phenotype in the ΔpptAB mutant strain is likely not associated with the SHP2, SHP3 quorum-sensing pathway in serotype M1 and M28 strains in this infection model.
Confirmation of the virulence role of glgP, a gene involved in carbohydrate utilization
Our TraDIS screens identified many GAS genes implicated in transport of nutrients such as amino acids, vitamins and carbohydrates. We next studied a gene likely to be involved in carbohydrate utilization. The GAS gene glgP was identified as essential for necrotizing myositis in the TraDIS screens conducted with both the serotype M1 and M28 transposon mutant libraries (Table S1). However, glgP has not previously been implicated in GAS virulence. glgP encodes an inferred protein with homology to E. coli glycogen phosphorylase (72). We generated isogenic mutant strain ΔglgP by deleting the glgP gene in serotype M1 strain MGAS2221. The ΔglgP isogenic mutant strain is severely attenuated in capacity to cause necrotizing myositis in NHPs, thereby confirming the TraDIS screen finding (Figure 8, A-D). We next evaluated the potential role of glgP in carbohydrate metabolism. Although the ΔglgP mutant strain has no growth defect in medium with glucose, this mutant strain has a severe growth defect when maltose or maltodextrin is provided as the sole carbohydrate in the culture medium (Figure 9, A-C). Consistent with the idea that the product of the glgP gene is involved in glycogen and starch utilization, bacteria grown in THY supplemented with starch showed evidence of starch accumulation in the isogenic mutant strain ΔglgP, but not the wild-type parental strain that retains the ability to metabolize starch (Figure 9D). Interestingly, in E. coli, glycogen accumulation is also significantly higher in glgP deletion mutants (72).
Expression of the GAS genes implicated in in vivo fitness genes during NHP necrotizing myositis
In the aggregate, data from the in vivo transposon mutant library screens and analysis of the isogenic mutant strains imply that the genes identified are expressed during NHP necrotizing myositis. To directly test for expression in vivo, we used TaqMan qRT-PCR to measure the transcript level of GAS transporter genes Spy0014, Spy0271, braB, Spy1726, pptA, and metabolic gene glgP in the NHP muscle tissue infected with M1 GAS MGAS2221. The transcript of all six of the GAS fitness genes studied were detectable by TaqMan qRT-PCR, thereby confirming that these genes are expressed in vivo in NHP necrotizing myositis (Figure 10A).
Expression of fitness genes in vivo in a human with necrotizing myositis
We next tested the hypothesis that the six targeted genes of interest are expressed in a human patient with necrotizing myositis. Necrotic skeletal muscle obtained from a patient with culture-proven GAS infection was studied by TaqMan qRT-PCR. The results confirmed the presence of transcripts from the six genes in the infected human patient (Figure 10B). Important to note, the relative transcript levels for all genes tested were closely similar in the experimentally infected NHPs and humans with natural infection.
Discussion
GAS is an abundant human pathogen that is responsible for substantial human illness and economic loss worldwide. Necrotizing fasciitis and myositis caused by this organism are particularly devastating infections because they have high morbidity and mortality. Effective treatment options for these infections remain limited and a licensed human GAS vaccine is not available. Thus, a fuller understanding of pathogen factors that contribute to these severe diseases is warranted and may facilitate translational research activities.
Our genome-wide screens identified 126 M1 genes and 116 M28 genes contributing to fitness in NHP necrotizing myositis. Of particular importance, we discovered a significant overlap between the genes identified in the M1 and M28 in vivo fitness screens, with 72 genes common to both serotypes, representing 57% and 64% of the M1 and M28 fitness genes, respectively. The similarity between M1 and M28 in vivo fitness gene results suggests the existence of conserved programs used by multiple diverse GAS strains to proliferate and damage tissue in necrotizing myositis. Many of the shared 72 genes encode proven or putative metabolic enzymes implicated in complex carbohydrate metabolism (malM and glgP), pyruvate metabolism (acoA, acoB, acoL), amino acid biosynthesis (tkt, aroD, glnA, and arcB), and nucleotide biosynthesis (purA and purB), suggesting these pathways are critical for GAS fitness in the environment of deep-tissue infection. In addition to metabolic genes, several previously identified GAS virulence or fitness factors were also among the 72 genes. For example, ScfAB (two putative membrane proteins) were identified as important for GAS fitness and virulence during subcutaneous infection in mice (32). adcABC (zinc importer) is critical for GAS virulence in mice and has vaccine interest (52).
In contrast to the similar critical gene requirements for serotype M1 and M28 GAS during experimental NHP necrotizing myositis, there is relatively little overlap between M1 GAS genes required for necrotizing myositis and growth in human saliva ex vivo (Figure 4). That is, the spectrum of genes contributing to fitness in these two environments is largely distinct. For example, genes encoding multiple amino acid transporters (e.g., Spy0014, braB and sstT) required for GAS fitness during necrotizing myositis were not identified as important for growth ex vivo in human saliva (Figure 4A). In contrast, a GAS phosphate transporter encoded by the pst operon is essential for persistence in human saliva ex vivo but is apparently dispensable for NHP muscle infection (Figure 4A). These results suggest that amino acid uptake is critical for GAS fitness during muscle infections, whereas phosphate uptake is essential for growth in human saliva ex vivo. Similarly, GAS genes contributing to NHP necrotizing myositis and mouse subcutaneous infection also have relatively little overlap (Figure 4B). Many GAS metabolic genes are specifically required for NHP necrotizing myositis. For example, genes for de novo purine nucleotide biosynthesis (purA and purB), carbohydrate utilization (glgP and malM), and arginine and citrulline catabolism (arcABCD) are uniquely important for NHP necrotizing myositis. Moreover, several known streptococcal virulence-modulating factors were also identified in NHP necrotizing myositis. These include genes for GAS lipoprotein processing (lgt and lsp) (73–75), and genes for a two-component regulatory system that is essential for GAS to evade human innate immunity (ihk and irr) (76, 77) (Figure 4B). Taken together, these results suggest the nutritional environment and the survival pressures present in the infected NHP skeletal muscle are distinct from those in human saliva ex vivo and in a mouse subcutaneous infection model. These results imply that complex gene programs used by GAS to cause other types of human infections (e.g., puerperal sepsis and pharyngitis) are also likely to be niche-specific.
A key theme of our M1 and M28 NHP genome-wide screens was identification of many genes encoding transporters that are required during necrotizing myositis. Pinpointing exactly which of the transporters contribute to bacterial virulence during necrotizing myositis sheds new light on the mechanisms of GAS-host interactions in this severe infection. We identified 13 distinct transporters that are required in both M1 and M28 GAS strains. Six of these transporters are inferred to function in amino acid transport. This observation suggests the ability to efficiently acquire host amino acids is critical for the pathogenesis of GAS necrotizing myositis. In vitro growth assays showed that amino acid transporter mutant strains ΔSpy0014, ΔbraB, and ΔSpy0271-0273 have a significant growth defect in the peptide-free CDM that is ameliorated by supplementing CDM with tryptone, a source of abundant peptides. These results suggest that efficient amino acid uptake is critical for wild-type GAS growth when the peptide source is limited, and the nutritional environment of the infected muscle is probably a poor source of available peptides. GAS is auxotrophic for 15 amino acids considered essential for growth (68). We hypothesized that transporters Spy0014, BraB and Spy0271-0273 are required for the highly efficient uptake of certain essential amino acids. As anticipated, we showed that supplementing CDM with methionine, histidine, and valine restored the growth of mutant strains ΔSpy0271-0273, ΔSpy0014 and ΔbraB, respectively, indicating these three transporters contribute to transport of these three amino acids. Interestingly, the concentration of free methionine, histidine, and valine in the human skeletal muscle tissue are 16.4 mg/L, 57.4 mg/L, and 30.5 mg/L, values lower than those present in CDM (100 mg/L) (78, 79). In the aggregate, our results suggest efficient uptake of essential host amino acids such as methionine, histidine, and valine is important for GAS to cause necrotizing myositis in NHPs. The qRT-PCR data demonstrating the presence of transcripts from these transporter genes document that they are expressed in NHPs and infected humans. Together, this implies that blocking the uptake of essential amino acids by GAS might be a feasible strategy to control GAS infection pathology, but further studies are required to test this idea.
We identified several virulence-related transporters with unclear functions. One example is the putative ABC transporter comprised of Spy1726 (transporter permease protein), Spy1727 (ATP-binding protein), and Spy1728 (substrate-binding lipoprotein). The TraDIS screen results and the virulence phenotype of the isogenic mutant strain indicate this ABC transporter of unknown function is critical for the ability of GAS to cause NHP skeletal muscle pathology. Although its function is not known, multiple additional leads indicate ABC transporter Spy1726-1728 plays a role in host-pathogen interactions. For example, the Spy1726-1728 operon is upregulated when GAS cells are in human blood and macrophages (80, 81). In addition, Spy1726-1728 is regulated by the CovR/S two-component system, a global virulence gene regulator in GAS (82). Proteomic studies show that the substrate–binding lipoprotein Spy1726 is located on the bacterial cell surface, and thus might be a candidate for vaccine or other translational research (70). Future structural and functional studies on this transporter appear to be warranted.
To summarize, in this work we used two distinct transposon mutant libraries made in serotypes of GAS that cause abundant human cases of severe invasive infections to identify genes contributing to GAS fitness in an NHP model of necrotizing myositis. NHP infection using six isogenic mutant strains confirmed the crucial requirement for the genes identified by our TraDIS screens. Our findings complement work conducted with other transposon mutant screens that identified GAS genes contributing to fitness during growth in vitro, in human saliva and human blood ex vivo, and mouse subcutaneous infection. The findings presented herein may ultimately lead to better ways to diagnose, treat, and prevent necrotizing myositis and fasciitis caused by GAS, infections with devastating consequences to the human host.
Methods
Bacterial strains
Strain MGAS2221 is genetically representative of the pandemic clone of serotype M1 GAS that arose in the 1980s and has spread worldwide (20). Strain MGAS27961 is genetically representative of a virulent serotype M28 clone that is prevalent in the United States and elsewhere (44). These two strains have wild-type alleles of all major transcriptional regulators known to affect virulence, such as covR and covS, ropB, mga, and rocA.
GAS strain growth conditions
The GAS strains were cultured in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY). When required, GAS strains were grown in a chemically defined medium (CDM) (79). Amino acids were added to the designated concentration. For growth in medium with a carbohydrate source other than glucose, GAS strains were cultured in maltose medium and maltodextrin medium. The composition of these media is presented in Table S8.
Transposon mutant libraries and culture conditions
The mutant library generated in serotype M1 strain MGAS2221 using transposon plasmid pGh9:ISS1 was recently described (31, 49). The serotype M28 strain MGAS27961 transposon mutant library was made by essentially identical methods. The strains were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY) broth at 37° C with 5% CO2.
Preparation of transposon mutant library frozen stock for nonhuman primate infection
100 µl of the stock transposon mutant library (M1 or M28 GAS library) was inoculated in 500 ml THY supplemented with 0.5 µg/ml erythromycin and cultured at 37° C for 8 hrs. The proliferated transposon library was pelleted by centrifugation, washed three times with saline, and then suspended in 10 ml saline supplemented with 20% glycerol. The suspended mutant library was aliquoted into cryogenic tubes and stored at −80° C until subsequent use in NHP infections.
Nonhuman primate necrotizing myositis infection model used for TraDIS analysis
A well-described NHP model of necrotizing myositis was used (18, 33). For the transposon mutant library screens, six cynomolgus macaques (1-3 years, 2-4 kg, males and females) each were used for the serotype M1 and M28 screens. Briefly, NHPs were sedated and bacteria were inoculated in the right quadriceps. Animals were observed and necropsied 24 hours post-infection. To analyze the output transposon insertion library, a ~0.5 g (~0.5-1.0 cm diameter) biopsy of necrotic muscle was obtained from the inoculation site, homogenized in 1 ml sterile PBS, transferred to 40 ml Todd Hewitt broth and incubated for 6 hrs. Before incubation, 100 µl were removed from the 40 ml culture, serially diluted in sterile PBS and plated to determine the number of CFUs in the output library.
Infected tissue was also collected for histologic examination. The inoculum used for the input M1 transposon mutant library was ~5×108 CFU/kg. The inoculum used for the input M28 transposon mutant library was 1×1010 CFU/kg. A higher dose of the M28 input transposon mutant library was used because in previous NHP studies, an approximately 10-to-100-fold higher inoculum of M28 strains compared to M1 strains was needed to generate similar disease character.
DNA preparation and massively parallel sequencing
The mutant library genomic DNA preparation and DNA sequencing were performed according to procedures described previously for TraDIS analysis (31, 49). The PCR-amplified libraries were sequenced with a NextSeq550 instrument (Illumina) using a single-end 75-cycle protocol.
Processing of TraDIS sequencing reads and data analysis
The processing of TraDIS reads and data analysis were performed according to previously described procedures (31). Briefly, the multiplexed raw Illumina reads obtained from the input and output mutant pools were parsed with FASTX Barcode Splitter (http://hannonlab.cshl.edu/fastx_toolkit/commandline.html). The resulting sequencing reads were analyzed with the TraDIS toolkit (83). tradis_comparison.R was used to compare the reads mapped per gene between the input pools (mutant libraries before NHP infection) and the output pools (mutant libraries recovered from infected NHP). The GAS genes with significantly changed mutant frequency (log2 fold-change greater than +/−1, and q value < 0.01) in the output mutant pools were interpreted as contributing to GAS fitness during NHP necrotizing myositis. Illumina sequencing reads of the M1 input library (n = 6), M1 output library (n = 6), M28 input library (n = 6), and M28 output library (n = 6) are deposited in the NCBI Sequence Read Archive (SRA) under the accession number (xxxxxxxxxx).
Construction and characterization of isogenic mutant strains
Isogenic mutant strains were derived from wild-type parental strain MGAS2221, the organism used for construction of the serotype M1 transposon mutant library. Primers used for generating the mutant strains are listed in Table S7. Markerless isogenic mutant strains were constructed by nonpolar deletion of the target gene(s) using allelic exchange (18). For example, to delete Spy0014, primer sets 0014-1/2 and 0014-3/4 were used to amplify two ~1.5 kb fragments flanking Spy0014 with genomic DNA purified from serotype M1 strain MGAS2221. The two flanking fragments were combined by overlap-extension PCR with primers 0014-1 and 0014-4. The combined fragment was cloned into suicide vector pBBL740 and transformed into parental strain MGAS2221. The plasmid integrant was used for allelic exchange as described previously (18). PCR was used to identify potential mutant candidates containing the desired deletion. All other isogenic mutant strains were generated using analogous methods. Whole genome sequencing of all isogenic mutant strains was done to confirm the absence of spurious mutations.
Infection of NHPs with isogenic mutant strains
To confirm the role of candidate genes in necrotizing myositis molecular pathogenesis and thereby validate the TraDIS data, the virulence of the parental wild-type strain MGAS2221 and the six isogenic deletion-mutant strains was assessed in the NHP necrotizing myositis infection model. Animals randomly assigned to different strain treatment groups received 108 CFU/kg of one strain (wild-type or isogenic mutant) in the right limb and a different strain in the left limb. Each strain was tested in triplicate. The animals were observed continuously and necropsied at 24 hrs post-inoculation.
Histopathology analysis
For histology evaluation, lesions were excised, and visually inspected. Lesions (necrotic muscle) were measured in three dimensions and volume was calculated using the formula for an ellipsoid. Tissue taken from the inoculation site was trisected, fixed in 10% phosphate buffered formalin, and embedded in paraffin using standard automated instruments. Histology of the three sections taken from each limb was scored by a pathologist blinded to the strain treatment groups as described previously (18, 19). To obtain the quantitative CFU data, diseased muscle obtained from the inoculation site or distal hip margin was weighed, homogenized (Omni International) in 1 mL PBS, and CFUs were determined by plating serial dilutions of the homogenate. Statistical differences between strain groups were determined using the Mann-Whitney test.
Iodine staining of wild-type and ΔglgP mutant strain
The wild-type strain and isogenic ΔglgP mutant strain were cultured for eight hours in 10 ml of THY supplemented with 2g/L of soluble starch (Sigma-Aldrich). GAS cells were pelleted and washed five times with saline to remove the culture medium. After the final saline wash, GAS cells were suspended in 1ml of saline. 10 µl of Gram’s iodine solution was added to the cell suspensions to visualize glycogen accumulation in the GAS cell. Only the ΔglgP mutant strain displayed a dark blue iodine stain phenotype.
Isolation of total RNA from GAS-infected non-human primates quadriceps muscle sections and skeletal muscle from a human with GAS necrotizing fasciitis
Infected tissue from NHPs or human patients was stored at −80°C in DNA/RNA Shield (Zymo Research) or RNAlater (Invitrogen), respectively, thawed on ice, transferred to a tube containing 2 ml of cold TE, and diluted with either 2 ml or 1.3 ml 2X DNA/RNA Shield. Tissue samples were homogenized with an Omni TH homogenizer (Omni International). Prior to lysis the supernatants were divided into either four aliquots each containing 900 µl, or 3 aliquots each containing 950 µl, for NHP or human samples, respectively. The tissues were lysed by ballistic disintegration using a FastPrep-96 instrument (MP Biomedicals) and Zymo tubes containing 0.1 and 0.5-mm ZR BashingBeads (Zymo Research). Lysis was repeated three times at 1,600 rpm for 1 min, and tubes were placed on ice for 1 min after each lysis step. Particulate matter present in the supernatants was eliminated with QIAshredder homogenizers (Qiagen). RNA was isolated using the Zymobiomics RNA kit (Zymo Research) following the manufacturer instructions with the exceptions that all aliquots from a particular sample were pooled together before passing them through the first column and the recommended DNase treatment was performed twice for the human samples. Total RNA quality was assessed with an RNA Nanochip and an Agilent 2100 Bioanalyzer (Agilent Technologies).
qRT-PCR analysis of infected NHP and human muscle
Total RNA extracted from infected NHP or human skeletal muscle was converted into cDNA using Superscript III reverse transcriptase, random primers, RNase OUT and dNTPs (all from Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using Taqman fast universal PCR master mix (Applied Biosystems) with an ABI 7500 Fast System instrument (Life Technologies). The genes tested were Spy0014, Spy0271, braB, Spy1726, pptA, and glgP. The sequences of primers and probes used in the qRT-PCR experiments are listed in the Table S9. Each experiment was performed with three technical replicates at three different dilutions. Transcript levels were normalized relative to the rpsL gene (encoding 30S ribosomal protein S12).
Statistics
Results of lesion volume and CFU recovery from NHPs are expressed as mean ± SEM, with statistically significant differences determined using the Mann-Whitney test (Prism 6, Graphpad Software). Results of histology scoring of infected NHP muscle are expressed as mean ± SEM, with statistically significant differences determined using the Wilcoxon Rank Sum Test (Prism 6). Nonparametric tests were used because the data were shown to not follow a normal distribution using the Shapiro-Wilk test (Prism 6).
Study approvals
All animal experiments were approved by the Institutional Animal Care and Use Committee of Houston Methodist Research Institute (protocol AUP-1217-0058). The human tissue was collected as part of a study approved by the Institutional Review Board at Houston Methodist Research Institute (protocol 0907-0151).
Author contributions
LZ performed and analyzed TraDIS experiments, constructed and characterized isogenic mutant strains and wrote the manuscript. RJO planned and conducted experiments involving the NHPs, analyzed resulting data and wrote the manuscript. SBB analyzed the genomic data and wrote the manuscript. JME contributed critical discussions about transporter physiology and performed the TaqMan qRT-PCR to measure the transcript level of GAS transporter genes in infected NHP and human muscle. MOS performed the genome sequencing of the isogenic mutant strains and provided technical support for the NHP studies. SLK constructed and characterized isogenic mutant strains. CCC provided extensive technical support for all phases of the study. ARLC and ASW provided intellectual guidance for the TraDIS data analysis. LJ oversaw and performed the NHP experiments. JMM designed the studies, analyzed experiments, wrote the manuscript and oversaw the project.
Supplemental material
Conflicts of interest
The authors have declared that no conflicts of interest exist.
Acknowledgments
This work was supported by funds from the Fondren Foundation (to James M. Musser). Amelia R. L. Charbonneau is supported by the University of Cambridge Doctoral Training Partnership scheme, which is funded by the Biotechnology and Biological Sciences Research Council, UK (reference 1503883). We thank Drs. Frank DeLeo, Magnus Gottsfredsson, Karl G. Kristinsson, David M. Morens, and Kathryn E. Stockbauer for critical reading of the manuscript and suggesting improvements. We are indebted to Dr. Lillian S. Kao for providing tissue specimens from a patient with necrotizing myositis. We thank Annessa Smith and Caroline White for superb veterinary technical assistance.
Reference
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