Creation of a functional S-nitrosylation site in vitro by single point mutation

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacement

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

A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence

Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC's, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS

A Forward Phenotypically Driven Unbiased Genetic Analysis of Host Genes That Moderate Herpes Simplex Virus Virulence and Stromal Keratitis in Mice

<div><p>Both viral and host genetics affect the outcome of herpes simplex virus type 1 (HSV-1) infection in humans and experimental models. Little is known about specific host gene variants and molecular networks that influence herpetic disease progression, severity, and episodic reactivation. To identify such host gene variants we have initiated a forward genetic analysis using the expanded family of BXD strains, all derived from crosses between C57BL/6J and DBA/2J strains of mice. One parent is highly resistant and one highly susceptible to HSV-1. Both strains have also been fully sequenced, greatly facilitating the search for genetic modifiers that contribute to differences in HSV-1 infection. We monitored diverse disease phenotypes following infection with HSV-1 strain 17syn+ including percent mortality (herpes simplex encephalitis, HSE), body weight loss, severity of herpetic stromal keratitis (HSK), spleen weight, serum neutralizing antibody titers, and viral titers in tear films in BXD strains. A significant quantitative trait locus (QTL) on chromosome (Chr) 16 was found to associate with both percent mortality and HSK severity. Importantly, this QTL maps close to a human QTL and the gene proposed to be associated with the frequency of recurrent herpetic labialis (cold sores). This suggests that a single host locus may influence these seemingly diverse HSV-1 pathogenic phenotypes by as yet unknown mechanisms. Additional suggestive QTLs for percent mortality were identified—one on Chr X that is epistatically associated with that on Chr 16. As would be anticipated the Chr 16 QTL also modulated weight loss, reaching significance in females. A second significant QTL for maximum weight loss in male and female mice was mapped to Chr 12. To our knowledge this is the first report of a host genetic locus that modulates the severity of both herpetic disease in the nervous system and herpetic stromal keratitis.</p></div

Staphylococcus aureus isolates encode variant staphylococcal enterotoxin B proteins that are diverse in superantigenicity and lethality.

Staphylococcus aureus produces superantigens (SAgs) that bind and cross-link T cells and APCs, leading to activation and proliferation of immune cells. SAgs bind to variable regions of the β-chains of T cell receptors (Vβ-TCRs), and each SAg binds a unique subset of Vβ-TCRs. This binding leads to massive cytokine production and can result in toxic shock syndrome (TSS). The most abundantly produced staphylococcal SAgs and the most common causes of staphylococcal TSS are TSS toxin-1 (TSST-1), and staphylococcal enterotoxins B and C (SEB and SEC, respectively). There are several characterized variants of humans SECs, designated SEC1-4, but only one variant of SEB has been described. Sequencing the seb genes from over 20 S. aureus isolates show there are at least five different alleles of seb, encoding forms of SEB with predicted amino acid substitutions outside of the predicted immune-cell binding regions of the SAgs. Examination of purified, variant SEBs indicates that these amino acid substitutions cause differences in proliferation of rabbit splenocytes in vitro. Additionally, the SEBs varied in lethality in a rabbit model of TSS. The SEBs were diverse in their abilities to cause proliferation of human peripheral blood mononuclear cells, and differed in their activation of subsets of T cells. A soluble, high-affinity Vβ-TCR, designed to neutralize the previously characterized variant of SEB (SEB1), was able to neutralize the variant SEBs, indicating that this high-affinity peptide may be useful in treating a variety of SEB-mediated illnesses

Quantitative trait loci for percent survival.

<p>Percent survival of male and female mice combined was employed to search for QTL associated with mortality. See the legend for fig. 2 for an explanation of the lines in the figure. A significant QTL was mapped to the distal end of Chr 16 and suggestive QTL were localized to Chr 4, 11, 12 and X (panel A). A pair scan analysis revealed a possible interaction between the locus on 16 and the suggestive locus on X (not shown). The marker regression function was used to identify SNPs with high LRS scores on Chr 16 and X, and the composite scan analysis was then employed to control for the Chr 16 or the Chr X locus. Controlling for the QTL on 16 (green arrow and line) revealed that the suggestive QTL on X was significant (panel B).</p

Schematic representation of the region of mouse Chr 16 from 75 to 90 mb.

<p>Suggestive (grey horizontal line) and significant (red horizontal line) LRS for HSV virulence (HSE) in male and female mice combined are indicated. The wavy blue line is the local LRS. The gold seismic graph along the X axis indicates local SNP density. The mouse susceptability QTL region is deliniated by a solid blue rectangle just above the SNP seismic graph. The region equivalent to the human cold sore frequency QTL is designated by a solid red rectangle just above the seismic graph at 75–82 mb. The location of the mouse equivalent (D16Ertd472e ) to the human “cold sore” gene (C21orf91) is indicated by a red triangle. A blue triangle indicates the 3’ end of the Grik1 gene that is an expression QTL for Grik1 in the nervous system. C21orf91 is a short gene shown as a gold box, which is not clearly discernable at this magnification (Top panel). Bottom panel. A three megabase region comprising the peak of the QTL is shown. Grik1, which is 395 kb in length is also shown as a gold rectangle and the other variously colored shapes indicate known or predicted genes and transcripts in the region. In GN, hovering the mouse cursor over the colored shapes (indicated by the open arrow in fig. 7, bottom) gives brief information about each gene. Clicking on the shapes calls up all related entries in the NCBI Gene database. The bands along the top are clickable and either increase the magnification, go to the respective region of the UCSC genome browser, or go to the respective region of the Ensemble genome browser. A fully functional and interactive figure can be generated and further explored on genenetwork.org for trait 16185 by performing the interval mapping function for Chr16.</p

Correlations between phenotypes that map to the distal end of Chr 16.

<p>Spearman Rank correlations were performed bewteen percent mortality and herpetic stromal keratitis datasets (left panel, p = 1.52×10⁻⁰⁵) or percent maximum weight loss of both male and female mice combined and percent mortality of male and female mice combined (right panel, p =  1.75×10⁻⁰⁶).</p