Host Genetic Variations and Sex Differences Potentiate Predisposition,Severity, and Outcomes of Group A Streptococcus-MediatedNecrotizing Soft Tissue Infections

Karthickeyan Chella Krishnan,a,b Santhosh Mukundan,b Jeyashree Alagarsamy,b Donna Laturnus,b Malak Kotbb

Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USAa; Department of Basic BiomedicalSciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota, USAb

Host genetic variations play an important role in several pathogenic diseases, and we previously provided strong evidence thatthese genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive group A Strepto-coccus (GAS) patients, including sepsis and necrotizing soft tissue infections (NSTIs). The goal of the present study was to inves-tigate how genetic variations and sex differences among four commonly used mouse strains contribute to variation in severity,manifestations, and outcomes of NSTIs. DBA/2J mice were more susceptible to NSTIs than C57BL/6J, BALB/c, and CD-1 mice,as exhibited by significantly greater bacteremia, excessive dissemination to the spleen, and significantly higher mortality. Differ-ences in the sex of the mice also contributed to differences in disease severity and outcomes: DBA/2J female mice were relativelyresistant compared to their male counterparts. However, DBA/2J mice exhibited minimal weight loss and developed smaller le-sions than did the aforementioned strains. Moreover, at 48 h after infection, compared with C57BL/6J mice, DBA/2J mice hadincreased bacteremia, excessive dissemination to the spleen, and excessive concentrations of inflammatory cytokines andchemokines. These results indicate that variations in the host genetic context as well as sex play a dominant role in determiningthe severity of and susceptibility to GAS NSTIs.

Streptococcus pyogenes or group A streptococci (GAS) are thecausative agents of a multitude of human diseases ranging

from nonsevere strep throat, pharyngitis, and impetigo to life-threatening necrotizing soft tissue infections (NSTIs) and strep-tococcal toxic shock syndrome (STSS) (1–12). The multifacetednature of invasive GAS infections requires several modes of patho-genic adaptation to evade host immune defenses to successfullycolonize and survive in host niches. In addition to the plethora ofpathogenic factors contributing to disease severity, variations inthe host genetic context play an equally important role in manip-ulating disease severity, manifestations, and outcomes.

The emergence of virulent strains of GAS bacteria in the early1980s coincided with a remarkable resurgence of severe and oftendeadly forms of invasive infections associated with STSS andNSTIs (5, 6, 12, 13). One particular strain that emerged duringthat time is the clonal M1T1 strain that disseminated globally andcontinues to be one of the most prevalently isolated strains frompatients with invasive and/or noninvasive GAS infections (12, 14–17). This strain produces many secreted and surface-bound viru-lence factors; most of them are adapted to evade human host de-fense mechanisms (12, 18). However, previous studies from ourlaboratory characterized indistinguishable, invasive M1T1 iso-lates from patients with starkly different disease severity and out-comes (19). These findings underscored the contribution of hostfactors to the stark differences in severity and outcomes of invasiveGAS infections.

Indeed, ensuing epidemiological studies of large cohorts ofinfected patients revealed that human leukocyte antigen (HLA)class II allelic variations contribute to important differences inthe severity of GAS sepsis by differentially presenting GAS su-perantigens (SAgs) to T-cell receptor (TCR) V� elements andthereby modulating host responses to SAgs that bind simulta-neously to TCR V� elements and to HLA class II molecules,

which are expressed on the surface of host cells. These hostresponses allow the interactions and exchange of intracellularsignals that elicit potent inflammatory responses (20–23). In-ability to contain these responses results in severe sepsis, sub-stantial morbidity, and, in many cases, death. Additional stud-ies of humanized HLA class II transgenic mice enabled us tocorroborate the role of SAgs in severe invasive GAS infectionsand revealed that genetic variations in host HLA class II mole-cules that present SAgs to T cells expressing variable TCR V�elements differentially potentiate severity, manifestations, andoutcomes of GAS sepsis (24, 25).

Ensuing studies using distinct, conventional, inbred mousestrains corroborated the contributions of variations in host ge-netic factors in manipulating the severity and outcomes of GASsepsis (26–28). However, because of the restricted genetic varia-tions in traditional inbred mouse strains, we used genetically di-verse panels of recombinant inbred BXD mouse strains whosegenetic variations resemble those seen in humans and that will

Received 18 September 2015 Returned for modification 25 October 2015Accepted 8 November 2015

Accepted manuscript posted online 16 November 2015

Citation Chella Krishnan K, Mukundan S, Alagarsamy J, Laturnus D, Kotb M. 2016.Host genetic variations and sex differences potentiate predisposition, severity,and outcomes of group A Streptococcus-mediated necrotizing soft tissueinfections. Infect Immun 84:416 –424. doi:10.1128/IAI.01191-15.

Editor: A. Camilli

Address correspondence to Malak Kotb, malak.kotb@med.und.edu.

K.C.K. and S.M. contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.01191-15.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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therefore be more likely to yield results that can be translated intoclinical practice and thus provide a better understanding of thepathogenesis of sepsis in patients (29, 30). By investigating thecontributions of host genetic variation to disease severity and out-comes and by using the robust translational model of geneticallydiverse BXD mouse strains, we mapped two quantitative trait locito mouse chromosomes 2 and X, identifying additional host ge-netic factors associated with the severity of GAS sepsis (29). Thesestudies revealed that in addition to genetic factors, other factors,including age, but not the sex or weight of the infected host, playan important role in modulating the susceptibility to and severityof GAS sepsis (30).

We undertook the present study to investigate whether varia-tion in host genetics would also modulate the severity and mani-festations of GAS-mediated NSTIs. To this end, we infected threeinbred strains and one outbred strain of mice with the clonalM1T1 strain of GAS and then analyzed inter-mouse strain varia-tion in the severity and outcomes of GAS NSTIs. We report herethat, among the strains tested, DBA/2J mice were most susceptibleto GAS NSTIs. In addition, mouse sex played a major role inpotentiating the susceptibility to and severity of NSTIs. For exam-ple, female DBA/2J mice were more resistant to NSTI than weremale DBA/2J mice. Furthermore, at 48 h after infection, DBA/2Jmice had greater bacteremia, greater bacterial dissemination tothe spleen, and increased concentrations of inflammatory cyto-kines and chemokines. Taken together, these findings underscorethe contributions of host genetic context and sex differences inmodulating host-mediated responses to GAS NSTIs and therebypotentiating differential susceptibility.

MATERIALS AND METHODSEthics statement. All animal experiments were approved by the Institu-tional Animal Care and Use Committee (IACUC) at the University ofNorth Dakota.

Bacteria and culture media. The representative M1T1 clonal GASisolate 5448WT (19) was routinely grown statically at 37°C in THY me-dium (Todd-Hewitt broth [Difco] supplemented with 1.5% [wt/vol]yeast extract) as described previously. Sheep blood agar (Becton Dickin-son, Franklin Lakes, NJ) was used as solid medium.

Animal studies. Age- and sex-matched, 8- to 12-week-old mice ofthree inbred strains (BALB/c, C57BL/6J, and DBA/2J) and one outbredstrain (CD-1) were infected subcutaneously with 5448WT; the dose permouse was 108 CFU in 100 �l of sterile phosphate-buffered saline (PBS).Control animals were injected with sterile PBS alone. After infection, eachmouse was housed in an individual cage to avoid contact with or influencefrom skin lesions of other infected mice. Animals were observed twice aday for a period of 7 days to identify those that died and to look forchanges in weight and skin lesions. Animals were humanely euthanized ifneeded and at specified time points (either 48 h after infection or duringthe 7-day infection timeline) for cardiac puncture to obtain blood forplasma separation and bacterial load estimations. Necrotic skin tissuesand spleen samples were collected and homogenized by a rotor statorhomogenizer (Omni International, Marietta, GA) for analysis of bacterialload and dissemination.

Cytokine analyses. Blood collected from DBA/2J and C57BL/6J mice48 h after infection with 5448WT was centrifuged at �2,000 � g for 3 minto remove the plasma. The concentrations of cytokines and chemokines inthese plasma samples were measured by using a Bio-Plex Pro Mouse Cy-tokine 23-Plex assay kit (Bio-Rad, Hercules, CA) according to the manu-facturer’s protocol.

Histological analyses. For histological analyses, at 48 h after infectionwith 5448WT, the necrotic skin tissues were excised from DBA/2J andC57BL/6J mice and immediately fixed in 10% neutral buffered formalinfor 24 h. The samples were then embedded in paraffin blocks, which wereprocessed to obtain 4-�m sections and stained with Mayer’s hematoxylin(Sigma-Aldrich, St. Louis, MO) and eosin (Leica Biosystems, Inc., Rich-mond, IL) staining system according to the manufacturer’s protocol. Thestained slides were examined using an EVOS FL AutoCell imaging system(Life Technologies, Grand Island, NY).

Statistical analyses. Statistical analyses were performed by Prismv6.0d (GraphPad, La Jolla, CA). Survival analyses were done with thelog-rank (Mantel-Cox) test. One-way analysis of variance (ANOVA) withBonferroni’s post hoc tests was used to evaluate differences in log-trans-formed bacterial load (skin, blood, and spleen) among the four strains.Log-transformed bacterial load differences between males and femaleswere examined by using unpaired, two-tailed t tests. Two-way ANOVAwith Bonferroni’s post hoc tests was used to evaluate differences in bothpercent weight change and change in lesion sizes among the four strainsover time. The critical significance value (�) was set at 0.05, and if the Pvalues were less than �, we reported that, by rejecting the null hypothesis,the observed differences were statistically significant.

FIG 1 Differential susceptibility to GAS due to host genetics and sex differences. Mice were infected subcutaneously with 108 CFU of GAS isolate 5448WT.Survival curves for the four strains C57BL/6J, DBA/2J, BALB/c, and CD-1 (a) and for C57BL/6J (b) and DBA/2J (c) males and females are shown. The datapresented are the percent survival (n � 20 for each strain or n � 10 for each sex from a total of three experiments). P values were calculated by log-rank(Mantel-Cox) tests. NS, not significant; *, P � 0.05; **, P � 0.01; ***, P � 0.001; ****, P � 0.0001.

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RESULTSDBA/2J mice are more susceptible to GAS NSTIs than the otherstrains tested. To investigate the ability of host genetics to influ-ence GAS NSTIs, we selected three inbred mouse strains (BALB/c,C57BL/6J, and DBA/2J) and one outbred mouse strain (CD-1)and infected each subcutaneously with equal doses of 5448WT

bacteria as described in Materials and Methods. Marked differ-ences in susceptibility (survival) were observed (Fig. 1a). DBA/2Jmice were more susceptible to GAS NSTIs than the other strains.The survival rate of DBA/2J mice was 15%, whereas the other threestrains—CD-1, BALB/c, and C57BL/6J— had survival rates of 90,75, and 72.7%, respectively. To identify whether there were differ-

FIG 2 Differential bacterial loads in response to GAS NSTIs. To enumerate bacterial loads and dissemination, we collected skin tissues, blood, and spleens fromsurviving and dead mice of strains C57BL/6J, DBA/2J, BALB/c, and CD-1 (a) and from strain C57BL/6J (b) and DBA/2J (c) males and females during the 7-dayinfection timeline as described in Materials and Methods. Each symbol represents one mouse, each horizontal bar denotes the mean value for each strain or sex,and the horizontal dotted line indicates the inoculum given. For dead mice for which we did not collect blood (�), the bacteremia counts were given an arbitraryvalue of 1010 CFU/ml (the value closest to the maximum bacteremia). The data presented are log-transformed bacterial loads (n � 20 for each strain or n � 10for each sex from a total of three experiments). P values were calculated using one-way ANOVA (among four strains) with Bonferroni’s post hoc analyses (amongC57BL/6J and DBA/2J mice) and unpaired, two-tailed t tests (between males and females of the indicated strains). NS, not significant; *, P � 0.05; **, P � 0.01;***, P � 0.001; ****, P � 0.0001.

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ences in bacterial load (in skin and blood) and bacterial dissemi-nation between the mouse strains, we analyzed blood, skin, andspleen samples from surviving and dead mice during the 7-dayinfection timeline as described in Materials and Methods. DBA/2Jmice had greater bacterial loads in the skin and blood and greaterbacterial dissemination to the spleen than did the three otherstrains of mice (Fig. 2a).

Resistant mice recover faster. We next analyzed whethermarked differences were seen in any additional comorbidities (i.e.,weight loss and lesion area) between the four strains. Mice began todie starting day 3 after infection. Mice that died had consistently lostweight, whereas resistant strains started gaining weight beyond 4 daysafter infection. Much to our surprise, compared to the other mousestrains, the susceptible DBA/2J mice lost minimal weight through day4 after infection. Nevertheless, there was a significant difference inweight loss between DBA/2J and the rest of the mouse strains, in

particular C57BL/6J (P � 0.0001), from days 2 to 4 after infection(Fig. 3a). Beyond day 4, the weight loss differences were not signifi-cant between DBA/2J and the other strains, and the lack of significantdifferences at this time may be due to the weight gain in the resistantstrains but not in DBA/2J mice (Fig. 3a). In contrast, lesion size dif-fered significantly between DBA/2J and C57BL/6J mice only on day 3after infection (Fig. 3d). Similar to the weight loss results, the NSTIlesions of susceptible DBA/2J mice were minimal in size compared tothose of the other strains until day 4 after infection. However, the sizeof the lesions in DBA/2J mice continually increased past day 4 afterinfection, whereas the size of lesions in resistant strains reached aplateau. In general, these observations revealed that the susceptiblemice continually lost weight and developed lesions of increasing size,whereas the resistant mice tended to recover more quickly.

Female DBA/2J mice are resistant to GAS NSTIs. To assesswhether sex has a role in mouse susceptibility to GAS NSTIs, the

FIG 3 Resistant groups heal faster. Shown are the percent weight changes and lesion sizes over the 7-day infection timeline for the four strains (a and d) and forC57BL/6J (b and e) and DBA/2J (c and f) male and female mice. Error bars indicate means � the standard errors of the means (SEM) (n � 20 for each strain orn � 10 for each sex from a total of three experiments). P values were calculated by two-way ANOVA, and if an interaction (P � 0.05) was revealed, thenBonferroni’s post hoc analyses were computed at different time points between C57BL/6J and DBA/2J (a and d) or between male and female mice of indicatedstrains (c and f). NS, not significant; *, P � 0.05; **, P � 0.01; ***, P � 0.001; ****, P � 0.0001.

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survival rates of male and female DBA/2J mice were compared.Among the susceptible DBA/2J mice, the percentage of survivingfemales was significantly greater than the percentage of survivingmales of the same strain (Fig. 1c). Further, we investigated theextent of bacterial load and dissemination within DBA/2J femalemice. They had lower bacterial loads along with less disseminationto the spleen as shown in Fig. 2c. Weight loss between females andmales differed significantly, with females losing minimal weightpast day 3 after infection (Fig. 3c). Similarly, DBA/2J female micehad relatively smaller lesions than did male mice (Fig. 3f). In con-trast, within one of the resistant strains (C57BL/6J), significantdifferences between the male and female mice were not observed(Fig. 1b, 2b, 3b, and 3e).

DBA/2J mice exhibit ineffective GAS clearance from theblood at 48 h after infection. We hypothesized that ineffectivebacterial clearance was responsible for the observed susceptibilityto NSTIs in DBA/2J mice. To test this hypothesis, we injected

equal doses of GAS subcutaneously into DBA/2J mice andC57BL/6J mice (one of the resistant strains). At 48 h after infec-tion, mice were bled, and samples of their infected skin andspleens were excised for analysis of bacterial load and dissemina-tion. Compared to C57BL/6J mice, DBA/2J mice had no signifi-cantly different bacterial load at the site of infection (skin), but thebacterial load in blood and dissemination to spleen in DBA/2Jmice were significantly greater than those in C57BL/6J mice (Fig.4a). Nevertheless, the female DBA/2J mice had no bacteria in theblood at 48 h after infection (Fig. 4b). These observations indicatethat the continual persistence of bacteremia due to ineffective bac-terial clearance might account for the increased dissemination andsubsequent death of DBA/2J mice. Despite the increased bactere-mia and dissemination, further analyses of weight loss and lesionarea revealed that DBA/2J mice had a significantly lower percent-age of weight loss and smaller lesions than did C57BL/6J mice at 48h after infection (Fig. 4b and c). Also, we did not observe any

FIG 4 DBA/2J mice do not efficiently clear bacteremia but still display minimal weight loss and smaller lesions than do C57BL/6J mice. At 48 h after infectionwith �108 CFU of GAS isolate 5448WT, skin tissue, blood, and spleen samples were collected and homogenized to enumerate bacterial loads and disseminationto the spleen in C57BL/6J and DBA/2J mice (a) and bacteremia levels in male and female mice of the DBA/2J strain (b). Each symbol represents one mouse, eachhorizontal bar denotes the mean value for each strain or sex, and the horizontal dotted line indicates the inoculum given. The data presented are log-transformedbacterial loads (n 19 for each strain or n � 9 for each sex from a total of three experiments). (c) The percent weight changes and lesion sizes in C57BL/6J andDBA/2J mice are shown. Error bars indicate means � the SEM (n 19 in each strain from a total of three experiments). P values were calculated by unpaired,two-tailed t tests for bacterial enumerations (a and b) and two-way ANOVA with Bonferroni’s post hoc analyses for weight loss and lesions (c). NS, not significant;*, P � 0.05; **, P � 0.01; ***, P � 0.001; ****, P � 0.0001.

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significant differences in weight loss or lesion size between maleand female DBA/2J mice at 48 h after infection (see Fig. S1 in thesupplemental material). These observations further corroboratethe results shown in Fig. 3.

DBA/2J mice produce greater concentrations of inflamma-tory cytokines and chemokines in response to GAS NSTIs thanC57BL/6J mice. We previously described host-dependent differ-ences in the levels of cytokine responses during severe invasive

GAS infections (21, 22, 24). To test whether the same scenario wasobserved in DBA/2J versus C57BL/6J mice, we analyzed theplasma recovered from these mice at 48 h after infection by usingmultiplex cytokine and chemokine analyses. As shown in Fig. 5,there was a significant increase in concentration in both inflam-matory cytokines (interleukin-6 [IL-6] and IL-12) and inflamma-tory chemokines (CXCL1/KC and CCL2/MCP-1) in DBA/2Jmice. However, the concentration of tumor necrosis factor alpha

FIG 6 No significant differences in cytokine and chemokine production between males and females of DBA/2J strain in response to GAS NSTIs. Shown are themeasured cytokine levels of IL-6 (a), IL-12 (b), KC (c), MCP-1 (d), and TNF-� (e) between GAS-infected male and female mice of DBA/2J strain at 48 h afterinfection. Error bars indicate the means � the SD (n � 9 for each sex from a total of three experiments). The P values were calculated by unpaired, two-tailed ttests. NS, not significant.

FIG 5 DBA/2J mice produce greater concentrations of inflammatory cytokines and chemokines than C57BL/6J mice. Shown are the results of the differentialanalyses of IL-6 (a), IL-12 (b), keratinocyte-derived cytokine (KC) (c), monocyte chemoattractant protein 1 (MCP-1) (d), and TNF-� (e) between GAS-infectedC57BL/6J and DBA/2J mice at 48 h after infection. Error bars indicate means � the SD (n 19 for each strain from a total of three experiments). P values werecalculated by unpaired, two-tailed t tests. *, P � 0.05; **, P � 0.01; ***, P � 0.001; ****, P � 0.0001.

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(TNF-�) was slightly higher in C57BL/6J mice (Fig. 5e). However,we did not see any significant differences in cytokine concentra-tions between male and female DBA/2J mice at 48 after infection(Fig. 6).

Female mice exhibit more adipose cells in response to GASNSTIs than male mice. A recent study has demonstrated that inresponse to Staphylococcus aureus skin infections in C57BL/6Jmice, dermal preadipocytes proliferate and protect against the in-fection (31). In addition, a previous study has shown that sex playsan important role in skin morphology and physiology (32). Basedon these studies, we hypothesized a similar proliferation againstGAS skin infections; also, we wanted to determine whether thereare any marked sex-based differences in this proliferation. To in-vestigate this, necrotic skin tissues excised from male and femalemice of DBA/2J and C57BL/6J strains at 48 h after infection withGAS bacteria were stained using hematoxylin and eosin. We ob-served that in response to GAS infection, female mice exhibitedmore adipocytes than their male counterparts (Fig. 7).

DISCUSSION

In this study, we demonstrate the novel finding that host ge-netic variations and sex differences predispose mice to GAS-mediated NSTIs. DBA/2J mice were relatively susceptible toGAS NSTIs: we observed increased mortality, ineffective bac-terial clearance, increased bacterial loads and dissemination,elevated inflammatory markers, and poor recovery from thecomorbidities of weight loss and lesions. Further, femaleDBA/2J mice were more resistant to GAS NSTIs than were malemice of the same strain. This finding underscores the necessityof incorporating differences in the host genetic context and sex

in studies investigating factors affecting host-pathogen inter-actions in GAS NSTIs.

Longitudinal and endpoint analyses of the comorbidities ofweight loss and lesion size revealed that these two are not reliablemarkers for disease progression. For instance, the most suscepti-ble DBA/2J mice lost minimal weight and developed smaller le-sions during the initial stages of infection, despite exhibitinggreater bacterial loads and dissemination and increased concen-trations of inflammatory cytokines and chemokines. Neverthe-less, continued loss of body weight and an increase in lesion sizewere observed in susceptible mice throughout the infection time-line.

Several investigators have successfully reproduced GAS NSTIsin mice by utilizing conventional inbred strains, including BALB/c(33, 34) and C57BL/6J (35, 36) mice, and outbred strains, such asCD-1 (37–39) and hairless Crl:SKH1-hrBR (40, 41) mice. How-ever, most of these studies utilized female mice alone; therefore,the underlying role of sex differences could not be examined inrelation to host genetic variations in differential susceptibility toGAS NSTIs. To our knowledge, none of the reported studies ofGAS NSTIs have utilized DBA/2J mice, although these mice havebeen used in the studies of GAS sepsis (26–30).

The role of host genetics and sex differences in GAS NSTIs hasnot been reported so far; however, the role of host genetics but notsex in GAS sepsis has been widely reported (22, 23, 26–30). Forexample, we previously documented that human leukocyte anti-gen (HLA) class II variations can contribute to differences in hostresponses to GAS SAgs (22, 23). Further, our application of asystems genetics approach to identify additional host factors that

FIG 7 Comparison of GAS-infected skin tissues from male and female mice of DBA/2J and C57BL/6J strains. At 48 h after infection with �108 CFU of GASisolate 5448WT, necrotic skin tissues excised from males and females of DBA/2J and C57BL/6J strains were stained with hematoxylin and eosin. Shown arerepresentative pictures of stained skin samples from DBA/2J (a to d) and C57BL/6J (e to h) male and female mice. Epidermis (E), dermis (D), and panniculuscarnosus (PC) are indicated. Scale bar, 200 �m.

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modulate GAS sepsis revealed that the IL-1� and prostaglandin Esynthase pathways are involved in this complicated pathogenesis(29, 30). Moreover, host contributions to pathogenesis of otherinfectious agents, including Ebola virus (42), herpes simplex virustype 1 (43), influenza H1N1 virus (44), and Burkholderia pseu-domallei (45), have also been widely studied. Taken together, wecan conclude that host-mediated pathogenesis has been a topic ofthorough investigation; however, to our knowledge, the contribu-tion of host genetic variations and sex differences to responses toGAS NSTIs in particular is a novel observation that we report herefor the first time.

In summary, the results presented here provide evidence forthe role of host genetics and sex in susceptibility to GAS NSTIs.Further in-depth systems genetics-based investigations, which arecurrently ongoing in our laboratory with the utilization of theadvanced recombinant inbred BXD panel, will provide a thor-ough understanding of the mechanism(s) by which host geneticsand other nongenetic cofactors (including sex, age, and bodyweight) may affect host-pathogen interactions in the context ofGAS NSTIs and should expose means by which to prevent and/orameliorate excessive morbidity and mortality associated withthese infections.

ACKNOWLEDGMENTS

We thank Kim Young for help in tissue sectioning and Nathan Riha forhelp in animal husbandry.

This study was supported by a startup grant (M.K.) from the Univer-sity of North Dakota. We have no conflicts of interest.

The funders had no role in study design, data collection and interpre-tation, or the decision to submit the work for publication.

FUNDING INFORMATIONA startup grant from the University of North Dakota provided funding toMalak Kotb. The funders had no role in study design, data collection andinterpretation, or the decision to submit the work for publication.

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