Nourtan F. Abdeltawab

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.

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.

Susceptibility to severe streptococcal sepsis: use of a large set of isogenic mouse lines to study genetic and environmental factors

Variation in responses to pathogens is influenced by exposure history, environment and the hosts genetic status. We recently demonstrated that human leukocyte antigen class II allelic differences are a major determinant of the severity of invasive group A streptococcal (GAS) sepsis in humans. While in-depth controlled molecular studies on populations of genetically well-characterized humans are not feasible, it is now possible to exploit genetically diverse panels of recombinant inbred BXD mice to define genetic and environmental risk factors. Our goal in this study was to standardize the model and identify genetic and nongenetic covariates influencing invasive infection outcomes. Despite having common ancestors, the various BXD strains (n strains=33, n individuals=445) showed marked differences in survival. Mice from all strains developed bacteremia but exhibited considerable differences in disease severity, bacterial dissemination and mortality rates. Bacteremia and survival showed the expected negative correlation. Among nongenetic factors, age – but not sex or weight – was a significant predictor of survival (P=0.0005). To minimize nongenetic variability, we limited further analyses to mice aged 40–120 days and calculated a corrected relative survival index that reflects the number of days an animal survived post-infection normalized to all significant covariates. Genetic background (strain) was the most significant factor determining susceptibility (P⩽0.0001), thus underscoring the strong effect of host genetic variation in determining susceptibility to severe GAS sepsis. This model offers powerful unbiased forward genetics to map specific quantitative trait loci and networks of pathways modulating the severity of GAS sepsis.

Development of a Murine Model for Aerosolized Ebolavirus Infection Using a Panel of Recombinant Inbred Mice

Countering aerosolized filovirus infection is a major priority of biodefense research. Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported. A murine model of aerosolized filovirus infection in mice should be useful for screening vaccine candidates and therapies. In this study, various strains of wild-type and immunocompromised mice were exposed to aerosolized wild-type (WT) or mouse-adapted (MA) Ebola virus (EBOV). Upon exposure to aerosolized WT-EBOV, BALB/c, C57BL/6 (B6), and DBA/2 (D2) mice were unaffected, but 100% of severe combined immunodeficiency (SCID) and 90% of signal transducers and activators of transcription (Stat1) knock-out (KO) mice became moribund between 7–9 days post-exposure (dpe). Exposure to MA-EBOV caused 15% body weight loss in BALB/c, but all mice recovered. In contrast, 10–30% lethality was observed in B6 and D2 mice exposed to aerosolized MA-EBOV, and 100% of SCID, Stat1 KO, interferon (IFN)-γ KO and Perforin KO mice became moribund between 7–14 dpe. In order to identify wild-type, inbred, mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 60 BXD (C57BL/6 crossed with DBA/2) recombinant inbred (RI) and advanced RI (ARI) mouse strains were exposed to aerosolized MA-EBOV, and monitored for disease severity. A complete spectrum of disease severity was observed. All BXD strains lost weight but many recovered. However, infection was uniformly lethal within 7 to 12 days post-exposure in five BXD strains. Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 90–100% lethality in two strains. Analysis of post-mortem tissue from BXD strains that became moribund and were euthanized at the lower dose of MA-EBOV, showed liver damage in all mice as well as lung lesions in two of the three strains. The two BXD strains that exhibited 90–100% mortality, even at a low dose of airborne MA-EBOV will be useful mouse models for testing vaccines and therapies. Additionally, since disease susceptibility is affected by complex genetic traits, a systems genetics approach was used to identify preliminary gene loci modulating disease severity among the panel BXD strains. Preliminary quantitative trait loci (QTLs) were identified that are likely to harbor genes involved in modulating differential susceptibility to Ebola infection.

Bioinformatics analysis of immune response to group A streptococcal sepsis integrating quantitative trait loci mapping with genome-wide expression studies

Individuals infected with genetically identical group Astreptococcal (GAS) strains develop starkly different dis-ease progression and outcome [1]. We reported that HLAclass II allelic variation contributes to differences in sys-temic disease severity by modulating host responses tostreptococcal superantigens [2]. Inasmuch as the bacteriaproduce additional virulence factors, we sought to iden-tify additional host gene networks modulating GAS sep-sis. Accordingly, we used two parallel approaches todefine these gene networks, quantitative trait loci (QTL)mapping and genome-wide transcriptome analyses. Tomap QTLs modulating response to severe GAS sepsis, weused advanced recombinant inbred (ARI) strains, whichare genetically diverse strains that have common ancestralparents [3]. We chose to use BXD strains of ARI mice, asparental strains C57Bl/6J (B6) and DBA/2J (D2) show dif-ferential response to GAS sepsis and BXD strains are heav-ily genotyped at 13377 SNPs and microsatellite markers.BXD strains, derived from B6 and D2 parental strains, arehomozygous inbred lines, each of which is genetically dis-tinct. Using 30 different BXD strains (n = 5–26 mice perstrain), we identified significant QTLs on chromosome 2that strongly modulate disease severity [4]. To narrowdown these mapped QTLs, we applied bioinformaticstools including: linkage, interval specific haplotype analy-ses, and gene ontology and we identified multiple candi-date gene networks modulating immune response tosepsis.As a parallel approach, we performed genome-wide tran-scriptome analyses comparing resistant and susceptiblestrains. This comparison revealed 93 genes that were dif-ferentially regulated in mice spleens 36 h post-infection.These genes belonged to gene networks involvingimmune response to sepsis; particularly notable exampleswere prostaglandin (Ptges) and interleukin1 (IL-1) familypathways. Quantitative expression analyses, using realtime PCR, of prostaglandin E synthase (

Chitosan and Sodium Alginate Combinations Are Alternative, Efficient, and Safe Natural Adjuvant Systems for Hepatitis B Vaccine in Mouse Model

Hepatitis B viral (HBV) infections represent major public health problem and are an occupational hazard for healthcare workers. Current alum-adjuvanted HBV vaccine is the most effective measure to prevent HBV infection. However, the vaccine has some limitations including poor response in some vaccinee and being a frost-sensitive suspension. The goal of our study was to use an alternative natural adjuvant system strongly immunogenic allowing for a reduction in dose and cost. We tested HBV surface antigen (HBsAg) adjuvanted with chitosan (Ch) and sodium alginate (S), both natural adjuvants, either alone or combined with alum in mouse model. Mice groups were immunized subcutaneously with HBsAg adjuvanted with Ch or S, or triple adjuvant formula with alum (Al), Ch, and S, or double formulations with AlCh or AlS. These were compared to control groups immunized with current vaccine formula or unadjuvanted HBsAg. We evaluated the rate of seroconversion, serum HBsAg antibody, IL-4, and IFN-γ levels. The results showed that the solution formula with Ch or S exhibited comparable immunogenic responses to Al-adjuvanted suspension. The AlChS gave significantly higher immunogenic response compared to controls. Collectively, our results indicated that Ch and S are effective HBV adjuvants offering natural alternatives, potentially reducing dose.

Resveratrol and Montelukast Alleviate Paraquat-Induced Hepatic Injury in Mice: Modulation of Oxidative Stress, Inflammation, and Apoptosis

Paraquat (PQ) is one of the most used herbicide worldwide. Its cytotoxicity is attributed to reactive radical generation. Resveratrol (Res) and montelukast (MK) have anti-inflammatory and antioxidant properties. The protective effects of Res, MK, or their combination against PQ-induced acute liver injury have not been investigated before. Therefore, we explored the protective potential of Res and/or MK against PQ hepatic toxicity in a mouse model. Mice were randomly assigned to five groups: group I served as the normal control and group II received a single dose of PQ (50 mg/kg, i.p.). Groups III, IV, and V received PQ plus oral Res (5 mg/kg/day), MK (10 mg/kg/day), and Res/MK combination, respectively. Res and/or MK reduced PQ-induced liver injury, evidenced by normalization of serum total protein, ALT, and AST. Res and/or MK significantly reversed PQ-induced oxidative stress markers glutathione and malondialdehyde. Res and/or MK significantly reduced PQ-induced inflammation reflected in TNF-α levels. Furthermore, Res and/or MK reversed PQ-induced apoptosis assessed by differential expression of p53, Bax, and Bcl-2. Histopathologic examination supported the biochemical findings. Although Res and MK displayed antioxidative, anti-inflammatory, and antiapoptotic activities, their combination was not always synergistic.

Meta-analysis of genes within QTLs of group A streptococcal sepsis and their expression QTLs reveal pathways modulating host differential response to streptococcal sepsis

Complex host–pathogen interactions modulate differential responses to group A streptococcal (GAS) sepsis systemic disease [1,2]. We previously found that host HLA-II allelic variations are associated with differential response to severe GAS sepsis [3]. In addition, using mouse models of GAS sepsis we found other host genetic factors contribute to disease severity by modulating inflammatory responses [4,5]. We applied systems genetics approaches and analyzed variations in disease severity phenotypes using advance recombinant inbred (ARI) BXD strain panel. We mapped quantitative trait loci (QTLs) associated with differential host response to severe GAS sepsis to mouse Chr2 and X [5]. The focus of the current study is to identify regulating genes within QTLs associated with differential GAS sepsis. To do so, we explored differences in expression and nsSNPs of genes within mapped QTLs using expression data sets of relevant tissues. We selected spleen, leukocytes and lung expression data sets deposited in GeneNetwork as most relevant data sets for GAS sepsis disease severity. Collectively, integration of QTL mapping of sepsis phenotypes with expression QTLs uncovered pathways that modulate differential susceptibility to severe GAS sepsis, underscoring the complexity of traits modulating severe GAS sepsis. Approaches used in our study provide a powerful, unbiased genetics approach for analyzing interactive traits modulating the outcomes of infectious diseases.