Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common γ-chain deficient NOD/LtSz-scid (NOD-scid IL2Rγnull) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rγnull mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rγnull mice with human HSC generate 6-fold higher percentages of human CD45+ cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rγnull mice contain human Ig+ B cells and lower numbers of human CD3+ T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4+CD8+ thymocytes as well human CD4+CD8− and CD4−CD8+ peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rγnull mice was validated by 1) high levels of TCR excision circles, 2) complex TCRβ repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rγnull mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.

The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS 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 pressure governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with 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)2,3 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 (NETs)5,6, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generate hypervirulent bacterial variants with increased risk of systemic dissemination. Keywords CMMB Disciplines Life Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences Publication DetailsThis article was originally published as Walker, MJ et al, DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection, Nature Medicine 13, 2007, 981- GAS is estimated to cause ~700 million cases of self-limited throat or skin infection each year worldwide 7 . Invasive GAS disease occurs in approximately 1/1,000 cases, with associated mortality of 25% 7 . Epidemic invasive disease is associated with the emergence of the globally disseminated GAS M1T1 clone 1,8 , which is distinguished from related strains by acquisition of prophages encoding virulence determinants such as superantigen SpeA and DNase Sda1 9,10 . In the M1T1 GAS clone, the transition from local to systemic infection can be linked to mutations in the two-component covRS regulator. The effect of these mutations is a distinct shift in the transcriptional 3 profile of invasive GAS isolates compared to mucosal (throat) isolates 3 . The covRS mutation and changes in gene expression are recapitulated upon subcutaneous challenge of mice and analysis of GAS disseminating to the spleen in comparison with those in the original inocolum 3 . Prominent changes in the transcriptional profile of invasive GAS isolates include a strong up-regulation of the DNase gene sda1, and a marked decrease in expression of the gene encoding the cysteine protease SpeB 3 .Sda1 is a virulence factor that protects GAS against neutrophil killing by degrading the DNA framework of NETs 5,6 . Abolishment of SpeB expression allows accumulation and activation of the broad spectrum host protease plasmin on the GAS bacterial surface 4 . A clinical correlation of GAS invasive disease severity and diminished SpeB expression has been established 2 .To elucidate the selection pressure for the rap...

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