Systems Genetics Approaches in Mouse Models of Group A Streptococcal Necrotizing Soft-Tissue Infections.

Mouse models are invaluable resources for studying the pathogenesis and preclinical evaluation of therapeutics and vaccines against many human pathogens. Infections caused by group A streptococcus (GAS, Streptococcus pyogenes) are heterogeneous ranging from mild pharyngitis to severe invasive necrotizing fasciitis, a subgroup of necrotizing soft-tissue infections (NSTIs). While several strains of mice including BALB/c, C3H/HeN, CBA/J, and C57BL/10 offered significant insights, the human specificity and the interindividual variations on susceptibility or resistance to GAS infections limit their ability to mirror responses as seen in humans. In this chapter, we discuss the advanced recombinant inbred (ARI) BXD mouse model that mimics the genetic diversity as seen in humans and underpins the feasibility to map multiple genes (genetic loci) modulating GAS NSTI. GAS produces a myriad of virulence factors, including superantigens (SAg). Superantigens are potent immune toxins that activate T cells by cross-linking T cell receptors with human leukocyte antigen class-II (HLA-II) molecules expressed on antigen-presenting cells. This leads to a pro-inflammatory cytokine storm and the subsequent multiple organ damage and shock. Inbred mice are innately refractive to SAg-mediated responses. In this chapter, we discuss the versatility of the HLA-II transgenic mouse model that allowed the biological validation of known genetic associations to GAS NSTI. The combined utility of ARI-BXD and HLA-II mice as complementary approaches that offer clinically translatable insights into pathomechanisms driven by complex traits and host genetic context and novel means to evaluate the in vivo efficiency of therapies to improve outcomes of GAS NSTI are also discussed.

Heterogeneity in FoxP3- and GARP/LAP-Expressing T Regulatory Cells in an HLA Class II Transgenic Murine Model of Necrotizing Soft Tissue Infections by Group A Streptococcus.

Invasive group A streptococcus (GAS) infections include necrotizing soft tissue infections (NSTI) and streptococcal toxic shock syndrome (STSS). We have previously shown that host HLA class II allelic variations determine the risk for necrotizing fasciitis (NF), a dominant subgroup of NSTI, and STSS by modulating responses to GAS superantigens (SAgs). SAgs are pivotal mediators of uncontrolled T-cell activation, triggering a proinflammatory cytokine storm in the host. FoxP3-expressing CD4+ CD25+ T regulatory cells (Tregs) comprise phenotypically and functionally heterogeneous subsets with a profound ability to suppress inflammatory responses. Specifically, activated Tregs, which express glycoprotein A repetitions predominant (GARP) and display latent transforming growth factor ß1 (TGF-ß1) complexes (latency-associated peptide [LAP]), exhibit strong immunosuppressive functions. The significance of Tregs that may participate in suppressing inflammatory responses during NSTI is unknown. Here, we phenotypically characterized FoxP3/GARP/LAP-expressing Tregs in GAS-infected or SAg (SmeZ)-stimulated splenocytes from transgenic (tg) mice expressing human HLA-II DRB1*15 (DR15 allele associated with nonsevere NF/STSS-protective responses) or DRB1*0402/DQB1*0302 (DR4/DQ8 alleles associated with neutral risk for combined NF/STSS). We demonstrated both in vivo and in vitro that the neutral-risk allele upregulates expression of CD4+ CD25+ activated effector T cells, with a significantly lower frequency of Foxp3+/GARP+ LAP- but higher frequency of Foxp3- LAP+ Tregs than seen with the protective allele. Additional in vitro studies revealed that the presentation of SmeZ by the neutral-risk allele significantly increases proliferation and expression of effector cytokines gamma interferon (IFN-γ) and interleukin-2 (IL-2) and upregulates CD4+ CD25+ T cell receptors (TCRs) carrying specific Vß 11 chain (TCRVß11+) T cells and Th1 transcription factor Tbx21 mRNA levels. Our data suggest that neutral-risk alleles may drive Th1 differentiation while attenuating the induction of Tregs associated with suppressive function.

Genetic Architecture of Group A Streptococcal Necrotizing Soft Tissue Infections in the Mouse.

Host genetic variations play an important role in several pathogenic diseases, and we have previously provided strong evidences that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive Group A Streptococcus (GAS) infections, including sepsis and necrotizing soft tissue infections (NSTIs). Our initial studies with conventional mouse strains revealed that host genetic variations and sex differences play an important role in orchestrating the severity, susceptibility and outcomes of NSTIs. To understand the complex genetic architecture of NSTIs, we utilized an unbiased, forward systems genetics approach in an advanced recombinant inbred (ARI) panel of mouse strains (BXD). Through this approach, we uncovered interactions between host genetics, and other non-genetic cofactors including sex, age and body weight in determining susceptibility to NSTIs. We mapped three NSTIs-associated phenotypic traits (i.e., survival, percent weight change, and lesion size) to underlying host genetic variations by using the WebQTL tool, and identified four NSTIs-associated quantitative genetic loci (QTL) for survival on mouse chromosome (Chr) 2, for weight change on Chr 7, and for lesion size on Chr 6 and 18 respectively. These QTL harbor several polymorphic genes. Identification of multiple QTL highlighted the complexity of the host-pathogen interactions involved in NSTI pathogenesis. We then analyzed and rank-ordered host candidate genes in these QTL by using the QTLminer tool and then developed a list of 375 candidate genes on the basis of annotation data and biological relevance to NSTIs. Further differential expression analyses revealed 125 genes to be significantly differentially regulated in susceptible strains compared to their uninfected controls. Several of these genes are involved in innate immunity, inflammatory response, cell growth, development and proliferation, and apoptosis. Additional network analyses using ingenuity pathway analysis (IPA) of these 125 genes revealed interleukin-1 beta network as key network involved in modulating the differential susceptibility to GAS NSTIs.

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

Host genetic variations play an important role in several pathogenic diseases, and we previously provided strong evidence that these genetic variations contribute significantly to differences in susceptibility and clinical outcomes of invasive group A Streptococcus (GAS) patients, including sepsis and necrotizing soft tissue infections (NSTIs). The goal of the present study was to investigate 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. Differences in the sex of the mice also contributed to differences in disease severity and outcomes: DBA/2J female mice were relatively resistant compared to their male counterparts. However, DBA/2J mice exhibited minimal weight loss and developed smaller lesions than did the aforementioned strains. Moreover, at 48 h after infection, compared with C57BL/6J mice, DBA/2J mice had increased bacteremia, excessive dissemination to the spleen, and excessive concentrations of inflammatory cytokines and chemokines. These results indicate that variations in the host genetic context as well as sex play a dominant role in determining the severity of and susceptibility to GAS NSTIs.

Bacterial superantigens promote acute nasopharyngeal infection by Streptococcus pyogenes in a human MHC Class II-dependent manner.

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as 'trademark' virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC -II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms.

Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice.

BACKGROUND: Shiga toxin (Stx)-producing E. coli (STEC) are responsible for foodborne outbreaks that can result in severe human disease. During an outbreak, differential disease outcomes are observed after infection with the same STEC strain. One question of particular interest is why some infected people resolve infection after hemorrhagic colitis whereas others progress to the hemolytic uremic syndrome (HUS). Host age and infection dose have been implicated; however, these parameters do not appear to fully account for all of the observed variation in disease severity. Therefore, we hypothesized that additional host genetic factors may play a role in progression to HUS. METHODS AND RESULTS: To mimic the genetic diversity in the human response to infection by STEC, we measured the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred (ARI) BXD panel. We first infected the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) with either 86-24 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. We found a significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 86-24. This observation suggested that a host factor that may be masked by Stx2a affects O157:H7 colonization in some genetic backgrounds. We then determined the TUV86-2 colonization levels of 24 BXD strains in the ICF model. We identified several quantitative trait loci (QTL) associated with variation in colonization by correlation analyses. We found a highly significant QTL on proximal chromosome 9 (12.5-26.7 Mb) that strongly predicts variation in colonization levels and accounts for 15-20 % of variance. Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL, and we identified five genes that are most likely responsible for the differential colonization. CONCLUSIONS: The identification of the QTL on chromosome 9 supports our hypothesis that individual genetic makeup affects the level of colonization after infection with STEC O157:H7.

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs.

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed "toolkits" utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.

Metal-mediated modulation of streptococcal cysteine protease activity and its biological implications.

Streptococcal cysteine protease (SpeB), the major secreted protease produced by group A streptococcus (GAS), cleaves both host and bacterial proteins and contributes importantly to the pathogenesis of invasive GAS infections. Modulation of SpeB expression and/or its activity during invasive GAS infections has been shown to affect bacterial virulence and infection severity. Expression of SpeB is regulated by the GAS CovR-CovS two-component regulatory system, and we demonstrated that bacteria with mutations in the CovR-CovS two-component regulatory system are selected for during localized GAS infections and that these bacteria lack SpeB expression and exhibit a hypervirulent phenotype. Additionally, in a separate study, we showed that expression of SpeB can also be modulated by human transferrin- and/or lactoferrin-mediated iron chelation. Accordingly, the goal of this study was to investigate the possible roles of iron and other metals in modulating SpeB expression and/or activity in a manner that would potentiate bacterial virulence. Here, we report that the divalent metals zinc and copper inhibit SpeB activity at the posttranslational level. Utilizing online metal-binding site prediction servers, we identified two putative metal-binding sites in SpeB, one of which involves the catalytic-dyad residues (47)Cys and (195)His. Based on our findings, we propose that zinc and/or copper availability in the bacterial microenvironment can modulate the proteolytic activity of SpeB in a manner that preserves the integrity of several other virulence factors essential for bacterial survival and dissemination within the host and thereby may exacerbate the severity of invasive GAS infections.

Streptococcal collagen-like protein A and general stress protein 24 are immunomodulating virulence factors of group A Streptococcus.

In Western countries, invasive infections caused by M1T1 serotype group A Streptococcus (GAS) are epidemiologically linked to mutations in the control of virulence regulatory 2-component operon (covRS). In indigenous communities and developing countries, severe GAS disease is associated with genetically diverse non-M1T1 GAS serotypes. Hypervirulent M1T1 covRS mutant strains arise through selection by human polymorphonuclear cells for increased expression of GAS virulence factors such as the DNase Sda1, which promotes neutrophil resistance. The GAS bacteremia isolate NS88.2 (emm 98.1) is a covS mutant that exhibits a hypervirulent phenotype and neutrophil resistance yet lacks the phage-encoded Sda1. Here, we have employed a comprehensive systems biology (genomic, transcriptomic, and proteomic) approach to identify NS88.2 virulence determinants that enhance neutrophil resistance in the non-M1T1 GAS genetic background. Using this approach, we have identified streptococcal collagen-like protein A and general stress protein 24 proteins as NS88.2 determinants that contribute to survival in whole blood and neutrophil resistance in non-M1T1 GAS. This study has revealed new factors that contribute to GAS pathogenicity that may play important roles in resisting innate immune defenses and the development of human invasive infections.

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection.

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.

Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone.

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.

Individual genetic variations directly effect polarization of cytokine responses to superantigens associated with streptococcal sepsis: implications for customized patient care.

Host immunogenetic variations strongly influence the severity of group A streptococcus sepsis by modulating responses to streptococcal superantigens (Strep-SAgs). Although HLA-II-DR15/DQ6 alleles strongly protect against severe sepsis, HLA-II-DR14/DR7/DQ5 alleles significantly increase the risk for toxic shock syndrome. We found that, regardless of individual variations in TCR-Vß repertoires, the presentation of Strep-SAgs by the protective HLA-II-DR15/DQ6 alleles significantly attenuated proliferative responses to Strep-SAgs, whereas their presentation by the high-risk alleles augmented it. Importantly, HLA-II variations differentially polarized cytokine responses to Strep-SAgs: the presentation of Strep-SAgs by HLA-II-DR15/DQ6 alleles elicited significantly higher ratios of anti-inflammatory cytokines (e.g., IL-10) to proinflammatory cytokines (e.g., IFN-γ) than did their presentation by the high-risk HLA-II alleles. Adding exogenous rIL-10 significantly attenuated responses to Strep-SAgs presented by the high-risk HLA-II alleles but did not completely block the response; instead, it reduced it to a level comparable to that seen when these superantigens were presented by the protective HLA-II alleles. Furthermore, adding neutralizing anti-IL-10 Abs augmented Strep-SAg responses in the presence of protective HLA-II alleles to the same level as (but no higher than) that seen when the superantigens were presented by the high-risk alleles. Our findings provide a molecular basis for the role of HLA-II allelic variations in modulating streptococcal sepsis outcomes and suggest the presence of an internal control mechanism that maintains superantigen responses within a defined range, which helps to eradicate the infection while attenuating pathological inflammatory responses that can inflict more harm than the infection itself.

Etiology of autistic features: the persisting neurotoxic effects of propionic acid.

BACKGROUND: Recent clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism. Propionic acid (PA) is a short chain fatty acid and an important intermediate of cellular metabolism. Although PA has several beneficial biological effects, its accumulation is neurotoxic. METHODS: Two groups of young Western albino male rats weighing about 45 to 60 grams (approximately 21 days old) were used in the present study. The first group consisted of oral buffered PA-treated rats that were given a neurotoxic dose of 250 mg/kg body weight/day for three days, n = eight; the second group of rats were given only phosphate buffered saline and used as a control. Biochemical parameters representing oxidative stress, energy metabolism, neuroinflammation, neurotransmission, and apoptosis were investigated in brain homogenates of both groups. RESULTS: Biochemical analyses of brain homogenates from PA-treated rats showed an increase in oxidative stress markers (for example, lipid peroxidation), coupled with a decrease in glutathione (GSH) and glutathione peroxidase (GPX) and catalase activities. Impaired energy metabolism was ascertained through the decrease of lactate dehydrogenase and activation of creatine kinase (CK). Elevated IL-6, TNFα, IFNγ and heat shock protein 70 (HSP70) confirmed the neuroinflammatory effect of PA. Moreover, elevation of caspase3 and DNA fragmentation proved the pro-apoptotic and neurotoxic effect of PA to rat pups CONCLUSION: By comparing the results obtained with those from animal models of autism or with clinical data on the biochemical profile of autistic patients, this study showed that the neurotoxicity of PA as an environmental factor could play a central role in the etiology of autistic biochemical features.

A novel study on amyloid ß peptide 40, 42 and 40/42 ratio in Saudi autistics.

OBJECTIVES: We examined whether plasma concentrations of amyloid beta (Aß) as protein derivatives play a central role in the etiology of autistic features. DESIGN AND METHODS: Concentrations of human Aß (1-42), Aß (1-40), and Aß (40/42) in the plasma of 52 autistic children (aged 3-16 years) and 36 age-matched control subjects were determined by using the ELISA technique and were compared. RESULTS: Compared to control subjects, autistic children exhibited significantly lower concentrations of both Aß (1-40) and Aß (1-42) and lower Aß (40/42) concentration ratio. Receiver operating characteristics curve (ROC) analysis showed that these measurements of Aß peptides showed high specificity and sensitivity in distinguishing autistic children from control subjects. CONCLUSIONS: Lower concentrations of Aß (1-42) and Aß (1-40) were attributed to loss of Aß equilibrium between the brain and blood, an imbalance that may lead to failure to draw Aß from the brain and/or impairment of ß- and γ- secretase's concentration or kinetics as enzymes involving in Aß production.

Systemic dysregulation of angiopoietin-1/2 in streptococcal toxic shock syndrome.

Streptococcal toxic shock syndrome (STSS) is characterized by diffuse vascular leak resulting from widespread endothelial activation. Angiopoietin-1 and -2 (Ang-1 and Ang-2), which are important regulators of endothelial quiescence and activation, respectively, are dysregulated in certain diseases that are associated with endothelial dysfunction, but they have not been previously investigated in STSS. Plasma Ang-1 and Ang-2 concentrations were measured in 37 patients with invasive streptococcal infection with and without concurrent STSS. Greater angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) occurred in STSS than in invasive infection without shock; dysregulation decreased with convalescence. These results suggest that systemic Ang-1 and Ang-2 dysregulation is associated with disease severity in invasive streptococcal infection and that plasma levels of Ang-1 and Ang-2 may serve as clinically informative biomarkers in STSS.

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections.

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.

Dissection of the molecular basis for hypervirulence of an in vivo-selected phenotype of the widely disseminated M1T1 strain of group A Streptococcus bacteria.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB(+)/SpeA(-)/Sda1(low) phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB(-)/SpeA(+)/Sda1(high) phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (DeltaCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.

Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice.

Despite the prevalence of H5N1 influenza viruses in global avian populations, comparatively few cases have been diagnosed in humans. Although viral factors almost certainly play a role in limiting human infection and disease, host genetics most likely contribute substantially. To model host factors in the context of influenza virus infection, we determined the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice and identified genetic elements associated with survival after infection. The lethal dose in these hosts varied by 4 logs and was associated with differences in replication kinetics and increased production of proinflammatory cytokines CCL2 and tumor necrosis factor alpha in susceptible DBA/2J mice. Gene mapping with recombinant inbred BXD strains revealed five loci or Qivr (quantitative trait loci for influenza virus resistance) located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus. In conjunction with gene expression profiling, we identified a number of candidate susceptibility genes. One of the validated genes, the hemolytic complement gene, affected virus titer 7 days after infection. We conclude that H5N1 influenza virus-induced pathology is affected by a complex and multigenic host component.

An unbiased systems genetics approach to mapping genetic loci modulating susceptibility to severe streptococcal sepsis.

Striking individual differences in severity of group A streptococcal (GAS) sepsis have been noted, even among patients infected with the same bacterial strain. We had provided evidence that HLA class II allelic variation contributes significantly to differences in systemic disease severity by modulating host responses to streptococcal superantigens. Inasmuch as the bacteria produce additional virulence factors that participate in the pathogenesis of this complex disease, we sought to identify additional gene networks modulating GAS sepsis. Accordingly, we applied a systems genetics approach using a panel of advanced recombinant inbred mice. By analyzing disease phenotypes in the context of mice genotypes we identified a highly significant quantitative trait locus (QTL) on Chromosome 2 between 22 and 34 Mb that strongly predicts disease severity, accounting for 25%-30% of variance. This QTL harbors several polymorphic genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, cocitation networks, and biological relevance, and identified interleukin1 alpha and prostaglandin E synthases pathways as key networks involved in modulating GAS sepsis severity. The association of GAS sepsis with multiple pathways underscores the complexity of traits modulating GAS sepsis and provides a powerful approach for analyzing interactive traits affecting outcomes of other infectious diseases.

Molecular requirements for MHC class II alpha-chain engagement and allelic discrimination by the bacterial superantigen streptococcal pyrogenic exotoxin C.

Superantigens (SAgs) are microbial toxins that bind to both TCR beta-chain variable domains (Vbetas) and MHC class II molecules, resulting in the activation of T cells in a Vbeta-specific manner. It is now well established that different isoforms of MHC II molecules can play a significant role in the immune response to bacterial SAgs. In this work, using directed mutational studies in conjunction with functional analyses, we provide a complete functional map of the low-affinity MHC II alpha-chain binding interface of the SAg streptococcal pyrogenic exotoxin C (SpeC) and identify a functional epitope in the beta-barrel domain that is required for the activation of T cells. Using cell lines that exclusively express individual MHC II isoforms, our studies provide a molecular basis for the selectivity of SpeC-MHC II recognition, and provide one mechanism by how SAgs are capable of distinguishing between different MHC II alleles.